Recombinant vaccines comprising immunogenic attenuated bacteria having RPOS positive phenotype

ABSTRACT

Attenuated immunogenic bacteria having an RpoS +  phenotype, in particular,  Salmonella enterica  serotype Typhi having an RpoS +  phenotype and methods therefor are disclosed. The Salmonella have in addition to an RpoS +  phenotype, an inactivating mutation in one or more genes which render the microbe attenuated, and a recombinant gene capable of expressing a desired protein. The Salmonella are attenuated and have high immunogenicity so that they can be used in vaccines and as delivery vehicles for genes and gene products. Also disclosed are methods for preparing the vaccine delivery vehicles.

This is a continuation-in-part of application Ser. No. 08/970,789, filedNov. 14, 1997, now U.S. Pat. No. 6,024,961.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to attenuated microbes and, moreparticularly, to novel attenuated bacteria having an RpoS⁺ phenotype foruse as vaccines and delivery vehicles for genes and gene products and tomethods for their preparation. This invention is particularly applicableto Salmonella such as Salmonella enterica serotype Typhi (also referredto as Salmonella typhi).

2. Description of the Related Art

Live attenuated Salmonella strains can serve as delivery vehicles forrecombinant antigens or other proteins. As antigen carriers, therecombinant Salmonella have been shown to be useful in live vaccines(For review see Curtiss et al. in Essentials of Musocal Immunology,Kagnoff and Kiyono, Eds., Academic Press, San Diego, 1996, pp. 599-611;Doggett and Brown, in Mucosal Vaccines, Kiyono et al., Eds., AcademicPress, San Diego, 1996 pp 105-118; see also Hopkins et al. Infect Immun.63:3279-3286, 1995; Srinavasin et al Vaccines 95, R. N. Chanock et al.,Eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y., p 273-280,1995).

Ideally, live attenuated vaccine strains should possess a balancebetween the two properties of attenuation and immunogenicity. Suchvaccine strains would not cause any disease or impair normal hostphysiology or growth, thus being attenuated, and at the same time beable to colonize the intestine and gut associated lymphoid tissue uponoral administration or other lymphoid organs upon administration by someother route so as to be immunogenic. As a practical matter, however,such an ideal balance has not been achieved (Curtiss, in New GenerationVaccines Woodrow and Levine, Eds., Marcel Dekker, Inc., New York, 1990,pp. 161-188). This may be a result of the almost exclusive focusing ofefforts in Salmonella vaccine development on improving the attenuationcomponent of strains rather than on producing strains with highimmunogenicity.

Work directed toward achieving attenuation in microbes for use invaccines has utilized attenuating mutations in biosynthetic genes,regulatory genes and/or genes involved in virulence. (See Doggett andBrown, supra). One such regulatory gene which has been mutated as ameans for achieving attenuation has been the rpoS gene. The rpoS geneencodes an alternative sigma factor, RpoS, which is known to regulatethe stationary phase expression of over 30 genes (for review, see Loewenand Hengge-Aronis, Annu Rev Microbiol 48:53-80, 1994). The rpoS gene hasbeen shown to contribute to the virulence of Salmonella entericaserotype Typhimurium (also referred to as Salmonella typhimurium) inmice by RpoS regulation of chromosomal as well as plasmid-borne genes(Fang et al., Proc Natl Acad Sci 89:11978-11982, 1992; Norel et al.,FEBS Microbiol Lett 99:271-276, 1992; Kowarz et al., J Bacteriol176:6852-6860, 1994). Similarly, RpoS is thought to contribute to thevirulence of Salmonella typhi in humans by an action on chromosomal genedeterminants of virulence, inasmuch as these microbes do not possess thevirulence plasmid present in S. typhimurium (Robbe-Saule et al., FEMSMicrobiol Let 126:171-176, 1995; Coynault et al., Mol Microbiol22:149-160, 1996). Mutant rpoS S. typhimurium strains have been shown tobe attenuated (Fang et al, supra) and capable of eliciting protectiveimmunity in mice (Nickerson and Curtiss, Abstracts of the 96th GeneralMeeting of the American Society for Microbiology B-141:179, 1996;Coynault et al., Mol Microbiol 22:149-160, 1996). As a result, it hasbeen suggested that rpoS mutants may be attractive candidates for thedevelopment of vaccines (Nickerson and Curtiss, supra).

Attenuated strains of Salmonella typhi have been used as human vaccinesagainst typhoid fever as well as against heterologous antigens when usedas recombinant antigen delivery vehicles (Forrest, in CRC Press Inc.,1994, pp. 59-80; Levine et al, in New Generation Vaccines Woodrow andLevine, Eds., Marcel Dekker, Inc., New York, 1990, pp. 269-287). Thesevaccines based upon Typhi strains have almost exclusively been derivedfrom the Ty2 strain, in particular, Ty21a, which contains a galEmutation along with other mutations. Ty2 and its Ty21a derivativevaccine strain have been shown to be rpoS mutants and this mutation mayaccount, at least in part, for the attenuation seen with Ty21a and withother vaccine strains derived from Ty2 presumably by the down regulationof chromosomal virulence genes controlled by the rpoS gene product. TheTy21a vaccine is typical of vaccines derived from Ty2 in that althoughbeing attenuated, the Ty21a vaccine has proven to have low vaccineefficacy, requiring three high doses of approximately 10¹⁰ cfu to induceprotective immunity in approximately two-thirds of the vaccinatedindividuals. (Forrest, supra). Thus, there remains a continuing need forSalmonella typhi strains which exhibit not only low virulence, but, alsohigh immunogenicity for use in vaccines suitable for the delivery of adesired gene product to a host.

Other strains of S. typhi have been reported which may, however, have afunctional rpoS gene although this was not appreciated at the time ofthe report. For example, human vaccines have been reported based uponthe 27V and ISP1820 strains (Reitman, J Infect Dis 117:101-107, 1967;Levine et al., J Infect Dis 133:424-429, 1976; Tacket et al., InfectImmun 60:536-541, 1992). Neither of these strains contained arecombinant gene nor were they used to deliver a recombinant gene in avaccine composition.

In a report of recombinant rpoS⁺ S. typhi, Coynault et al. disclosed theconstruction of a Ty2 derivative containing a recombinant rpoS genewhich gave the microbe an RpoS⁺ phenotype. However, this Ty2 derivativewas used only in a laboratory study and no additional recombinant genewas incorporated nor was there any teaching of the use of thisderivative in a vaccine composition.

Finally, the S. typhi strains ISP1820 and ISP1822 (U.S. Pat. Nos.5,387,744 and 5,294,441 and PCT application WO/9424291) and the S. typhistrain 531Ty (U.S. Pat. No. 4,837,151) have been used to constructderivative vaccine strains. Although the studies reported herein showISP1820, ISP1822 and 531Ty to be RpoS⁺, this was not known at the timeof these earlier publications. Furthermore, none of these referencesrecognized the importance of the presence of a functional rpoS gene inachieving a high immunogenicity in a vaccine preparation. As a result,these references did not disclose the selection of vaccine strains basedupon the presence of an RpoS⁺ phenotype.

All references cited in this specification either supra or infra arehereby incorporated by reference. The discussion of the referencesherein is intended to summarize the assertions made by their authors andno admission is made as to the accuracy or pertinency of the citedreferences or that any reference is material to patentability.

SUMMARY OF THE INVENTION

In accordance with the present invention, the inventors herein havesucceeded in discovering the critical importance of a functional rpoSgene in Salmonella vaccine strains in that the presence of a functionalrpoS gene and an RpoS⁺ phenotype confers upon the Salmonella theproperty of high immunogenicity. As a result, when the RpoS⁺ phenotypeis present with one or more inactivating mutations other than a mutationin an rpoS gene, which render the microbe attenuated, a new andadvantageous balance of attenuation and high immunogenicity is achieved.This invention is particularly applicable to S. typhi based vaccines,however, it is also applicable to other Salmonella such as S. paratyphiA, B and C as well as to other serotypes of S. enterica such asTyphimurium, Enteritidis, Dublin and Choleraesuis. The invention is alsoapplicable to other bacteria having an rpoS gene, or functionalequivalent thereof, that can colonize human tissues, including Shigella,E. coli, and hybrids between such bacteria, such as Salmonella-Shigellahybrids, Salmonella-E. coli hybrids or Shigell-E. coli hybrids.

In one embodiment of the present invention, a method is provided fordelivery of a desired gene product to a human. The method comprisesselecting a strain of bacteria such as S. typhi on the basis of thestrain having (i) an RpoS⁺ phenotype, (ii) one or more inactivatingmutations which render the strain attenuated, and (iii) a recombinantgene encoding the gene product. The selecting step with respect to RpoS⁺phenotype can involve, in whole or in part, testing the strain todetermine its RpoS phenotype. The strain thus selected is thenadministered to the human. The one or more inactivating mutations whichrender the strain attenuated can involve a mutation in one gene or amutation in each of two or more genes.

The RpoS⁺ phenotypic activities of the Salmonella or other bacteria canbe produced by a chromosomal rpoS gene and/or by a recombinant.geneintroduced into the strain. Thus, in another embodiment, the methodcomprises administering to a human a live attenuated strain of bacteriahaving (a) an RpoS⁺ phenotype, (b) a recombinant rpoS⁺ gene, (c) one ormore inactivating mutations which render said microbe attenuated and (d)a second recombinant gene encoding the desired product. By recombinantrpoS⁺ gene or wild-type rpoS gene it is meant that the rpoS gene iscapable of producing a functional rpoS gene product. The termrecombinant rpoS⁺ gene is intended to refer to an rpoS gene introducedinto a microbe by human intervention and to exclude an rpoS genetransferred from and by a wild-type microbe using a natural means ofgene transfer such as conjugation, transduction or transformation,without aid of human intervention.

The attenuated microbes of the present invention contain at least onerecombinant gene capable of expressing a desired gene product, whichallows their use as carriers or delivery vehicles of the gene product tohumans. Examples of gene products deliverable by the microbes of theinvention include but are not limited to: antigens, which can be from ahuman pathogen, or, for use in autoimmune applications, from the humanitself, such as, for example, a gamete-specific antigen; enzymes thatcan synthesize antigens such as polysaccharides, lipoproteins,glycoproteins, and glycolipids; allergens of the human; immunoregulatorymolecules; hormones; and pharmacologically active polypeptides. Bydelivery of the desired gene product it is meant that either the geneproduct or the polynucleotide, i.e. nucleic acid, either DNA or RNA,encoding the product is delivered to the human. In embodiments in whichthe attenuated bacteria contains a recombinant rpoS gene, the desiredgene product is encoded by a second recombinant gene.

In another embodiment, the present invention provides a method forproducing a strain of carrier microbes for delivery of a desired geneproduct to a human. The method comprises (1) selecting for a strain ofS. typhi or other bacteria having an RpoS⁺ phenotype; (2) producing oneor more inactivating mutations in the RpoS⁺ strain to render the strainattenuated; and (3) introducing into the strain a recombinant geneencoding a desired gene product. The selecting step can involve, inwhole or in part, testing the strain to determine its RpoS phenotype.Steps 1-3 can be performed in any order.

In a further embodiment, the present invention involves another methodfor producing carrier microbes for delivery of a desired gene product toa human. The method comprises generating a live attenuated strain of S.typhi or other bacteria having (a) an RpoS⁺ phenotype, (b) a recombinantrpoS⁺ gene, (c) one or more inactivating mutations which render saidmicrobe attenuated and (d) a second recombinant gene encoding thedesired product.

Another embodiment of the present invention provides a carrier microbefor the delivery of a desired gene product to a human. The microbecomprises a live attenuated strain of S. typhi or other bacteria having(a) an RpoS⁺ phenotype, (b) a recombinant rpoS⁺ gene, (c) one or moreinactivating mutations which render said microbe attenuated and (d) asecond recombinant gene encoding the desired product.

In another embodiment a vaccine is provided for immunization of a human.The vaccine comprises a live attenuated strain of S. typhi or otherbacteria having (a) an RpoS⁺ phenotype, (b) a recombinant rpoS⁺ gene,(c) one or more inactivating mutations which render said microbeattenuated and (d) a second recombinant gene encoding the desiredproduct.

The present invention also provides in another embodiment, a geneticallyengineered cell. By genetically engineered cell reference is made to acell in which the DNA has been manipulated in vitro by humanintervention, for example, by gene splicing, to generate a newcombination of genes, to place a given gene or genes under the controlof a different regulatory system, to introduce specific mutations intothe DNA molecule and the like. The generation of the geneticallyengineered cells can employ any combination of molecular genetic andclassical microbial genetic means to effect construction of thegenetically engineered cell.

The genetically engineered cell comprises a live attenuated strain of S.typhi or other bacteria having (a) an RpoS⁺ phenotype, (b) a recombinantrpoS⁺ gene, (c) one or more inactivating mutations which render saidmicrobe attenuated and (d) a second recombinant gene encoding thedesired product. A method is also provided for the preparation of avaccine comprising mixing the genetically engineered cells with apharmaceutically acceptable formulation suitable for administration to ahuman.

The present invention also provides a genetically engineered bacteria,in particular S. typhi, containing a recombinant virulence gene that isregulated by RpoS, or its functional equivalent, in wild-type bacteriaand a method for using the genetically engineered bacteria for thedelivery of a desired gene product to a human. The recombinant virulencegene is capable of expressing a gene product that facilitates invasionand colonization of any of the gut associated lymphoid tissues (GALT),nasal associated lymphoid tissue (NALT) or the bronchial associatedlymphoid tissue (BALT) and the like which can collectively be called themucosal associated lymphoid tissue (MALT). The genetically engineered S.typhi or other bacteria can be further characterized as having one ormore inactivating mutations which render the microbe attenuated as wellas a second recombinant gene encoding the desired product.

In still another embodiment, the present invention provides a method forassessing the RpoS phenotype as an indication of the immunogenicity of abacteria strain, and in particular, of a Salmonella. It is believed thatmany bacterial strains propagated and maintained under laboratoryconditions accumulate ropS mutations. Thus, it would be useful toprovide a method for assessing the RpoS phenotype of a Salmonella orother bacteria, particularly for a strain being developed for use in avaccine. The method comprises determining the RpoS phenotype of thebacteria by assessing characteristics of the microbe regulated by RpoS.An increased immunogenicity is indicated by the presence of an RpoS⁺phenotype compared to the immunogenicity of an isogenic strain having anRpoS⁻ phenotype. The isogenic RpoS⁻ strain does not exhibit an RpoS⁺phenotype, but otherwise has the same genetic background as the teststrain.

As noted above, the delivery of a polynucleotide encoding the desiredgene product to a human is within the scope of the methods andcompositions.of the present invention. Moreover, each of the embodimentsabove involving methods and compositions based upon microbes having anRpoS⁺ phenotype are further contemplated to include methods andcompositions for the delivery of a gene or portion thereof to the cellsof a human. The gene or portion thereof can comprise a eukaryoticexpression cassette that contains the genetic information, either DNA orRNA, that is intended to be delivered to cells of the human.

Thus, in one embodiment of the present invention provides methods fordelivery of a gene or portion thereof to the cells of a human. One suchmethod comprises selecting a strain of bacteria such as S. typhi on thebasis of the strain having (i) an RpoS⁺ phenotype, (ii) one or moreinactivating mutations which render the strain attenuated, and (iii) thegene or portion thereof. The gene or portion thereof can be within aeukaryotic expression cassette. The selecting step with respect to RpoS⁺phenotype can involve, in whole or in part, testing the strain todetermine its RpoS phenotype. The method can also comprise delivering tocells of a human, a live attenuated strain of bacteria having (a) anRpoS⁺ phenotype, (b) a recombinant rpoS⁺ gene, (c) one or moreinactivating mutations which render said microbe attenuated and (d) thegene or portion thereof. The gene or portion thereof can be within aeukaryotic expression cassette.

The present invention also provides methods for producing a strain ofcarrier microbes for delivery of a desired gene or portion thereof to acell of a human. One such method can comprise (1) selecting for a strainof S. typhi or other bacteria having an RpoS⁺ phenotype; (2) producingone or more inactivating mutations in the RpoS⁺ strain to render thestrain attenuated; and (3) introducing into the strain the gene orportion thereof. The gene or portion thereof can be within a eukaryoticexpression cassette. The selecting step can involve, in whole or inpart, testing the strain to determine its RpoS phenotype and the stepscan be performed in any order. The method can also comprise generating alive attenuated strain of S. typhi or other bacteria having (a) an RpoS⁺phenotype, (b) a recombinant rpoS⁺ gene, (c) one or more inactivatingmutations which render said microbe attenuated and (d) the desired geneor portion thereof. The gene or portion thereof can be within aeukaryotic expression cassette.

The bacteria that can be used for delivery of a gene or portion thereofcan be an attenuated Salmonella, E. coli or Shigella orSalmonella-Shigella hybrid, Salmonella-E. coli hybrid or Shigella-E.coli hybrid so long as the attenuated bacteria releases the nucleic acidwithin the target host cell.

Among the several advantages achieved by the present invention,therefore, may be noted the provision of a carrier microbe which iscapable of colonizing and delivering a desired gene product or a desiredpolynucleotide to the gut associated lymphoid tissue if administeredorally, to the nasal associated lymphoid tissue if administeredintranasally and to other lymphoid organs if administered by otherroutes; the provision of an efficient and inexpensive method fordelivery of a nucleic acid molecule to human cells based upon the use ofRpoS⁺ carrier bacteria cells that release the nucleic acid molecule; theprovision of vaccine preparations which are highly immunogenic alongwith being attenuated; the provision of methods of delivering a desiredgene product or polynucleotide to an individual by administering thecarrier microbe so as to elicit an immune response; the provision ofmethods of preparing RpoS⁺ carrier microbes and vaccines wherein thevaccines are not only attenuated but also have high immunogenicity; andthe provision of methods for assessing the immunogenicity of aSalmonella or other bacteria by determining its RpoS phenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the time course of survival within J774 murinemacrophage-like cells of an rpoS⁺ Salmonella typhimurium, χ3339, and anisogenic rpoS mutant Salmonella typhimurium, χ4973.

FIG. 2 illustrates the time course of survival within rat bone marrowderived macrophages of an rpoS⁺ Salmonella typhimurium, χ3339, and anisogenic rpoS mutant Salmonella typhimurium, χ4973.

FIG. 3 illustrates light micrographs at approximately 200× magnification(indicated by a 50 μm bar) showing normal murine Peyer's patch tissue inFIG. 3A; murine Peyer's patch tissue at one day (FIG. 3B), three days(FIG. 3C), and five days (FIG. 3D) after peroral infection with χ4973;and murine Peyer's patch tissue after peroral infection with χ3339 atone day (FIGS. 3E and 3F), three days (FIG. 3G), and five days (FIG. 3H)post infection.

FIG. 4 illustrates transmission electron micrographs at approximately2000× magnification (indicated by a 5 μm bar) showing normal murinePeyer's patch lymphoid tissue (FIG. 4A), and murine Peyer's patchlymphoid tissue at five days after peroral infection with χ4973 (FIG.4B) or χ3339 (FIG. 4C).

FIG. 5 illustrates transmission electron micrographs at approximately2000× magnification (indicated by a 50 μm bar) showing normal murinePeyer's patch tissue (FIG. 5A), and normal murine Peyer's patch tissuefive days after peroral infection with χ4973 (FIG. 5B) or χ3339 (FIG.5C).

FIG. 6 illustrates the construction of plasmid vectors and bacterialstrains with the defined ΔphoPQ23 mutation.

FIG. 7 illustrates the construction of plasmid vectors and bacterialstrains with the defined ΔasdA16 mutation.

FIG. 8 illustrates the expression of the recombinant HBV core-pre-Sprotein by S. typhimurium Δcya Δcrp Δasd RpoS⁺ and RpoS⁻ strainscontaining Asd⁺ vector, pYA3167, expressing the HBV core-pre-S antigenconstructed by introducing the Asd⁺ vector into S. typhimurium χ8296(Acys Acrp Aasd) and χ8309 (Δcys Δcrp Δasd rpoS) examined by(A)Coomassie blue stained 12% sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) and (B) Western blot with amonoclonal antibody directed to the preS2 epitope with lanes in bothgels as follows: lane 1, molecular markers; lane 2, χ8296 (Δcya-27Δcrp-28 ΔasdA16 cfs RpoS⁺); lanes 3 & 4, χ8296 containing pYA3167 (Asd⁺vector expressing HBV core-pre-S Ag); lane 5, χ8309 (Acya-27 Acrp-28ΔasdA16 cfs ropS); lanes 6 and 7, χ8309 plus pYA3167 (Asd⁺ vectorexpressing HBV core-pre-S Ag).

FIG. 9 illustrates the induction of antibody titers to HBV core-pre-Sprotein expressed by S. typhimurium, SL1344 strains in which mice wereorally immunized with 10⁹ CFU or χ8296 (Δcya Δcrp Δasd RpoS⁺) containingpYA3167 (Asd⁺ vector specifying HBV core-pre-S) or the correspondingRpoS⁻ derivative, χ8309 (Δcya Acrp Δasd RpoS⁺ containing pYA3167)showing (A) serum IgG antibody titer and (B) IgA antibody in vaginalwashings determined at 4 and 6 weeks after immunization by ELISA using arecombinant polypeptide representing the full length pre-S sequence as acoating antigen (n=4).

FIG. 10 illustrates the levels of serum IgG antibodies to (A)recombinant HBV pre-S protein and (B) typhimurium LPS in groups of 8mice perorally immunized with 2 doses of 10⁹ CFU of the RpoS⁺ strain,χ8296(pYA3167), (open circles) or the RpoS⁻ strain, χ8309(pYA3167),(open squares) and 10⁸ CFU of RpoS⁺ strain, χ8296(pYA3167), (solidcircles) or the RpoS⁻ strain, χ8309(pYA3167), (solid squares) whereinserum samples were collected on weeks 2, 4, 6, and 8 weeks after dosing,pooled (N=8), diluted 1:400, measured by ELISA and expressed in thefigure as optical density at 405 nm.

FIG. 11 illustrates the levels of serum IgA antibodies to (A)recombinant HBV pre-S protein and (B) typhimurium LPS in groups of 8mice perorally immunized with 2 doses of 10⁹ CFU of the RpoS⁺ strain,χ8296(pYA3167), (solid bars) or the RpoS⁻ strain, χ8309(pYA3167),(hatched bars) wherein serum samples were collected on weeks 2, 4, 6,and 8 weeks after dosing, pooled (N=8), diluted 1:400, measured by ELISAand expressed in the figure as optical density at 405 nm.

FIG. 12 illustrates the pYA3433 plasmid.

FIG. 13 illustrates the pYA3467 plasmid.

FIG. 14 illustrates Coomassie staining of 12% sodium dodecyl sulfate(SDS), polyacrylamide gel electrophoresis (PAGE) to show expression ofthe recombinant hybrid HBcAg-pre-S antigen in S. typhi ΔphoPQ Δasdvaccine strains, wherein the arrow indicates the position of therecombinant antigen for lane 1, polypeptide SDS-PAGE size standards;lane 2, MGN-1191; lane 3, MGN-1191/pYA3167, transformant #1 (χ8281);lane 4, MGN-1191/pYA3167, transformant #2; lane 5, MGN-1191/pYA3167,transformant #3; lane 6, MGN-1256; lane 7, MGN-1256/pYA3167,transformant #1 (χ8280); lane 8, MGN-1256/pYA3167, transformant #2; lane9, MGN-1256/pYA3167, transformant #3; and lane 10, χ621/pYA3167.

FIG. 15 illustrates immunostaining with anti-HBV-preS monoclonalantibody following SDS-12% PAGE to show expression of the recombinanthybrid HBcAg-pre-S antigen in S. typhi ΔphoPQ Δasd vaccine strains,wherein the arrow indicates the position of the recombinant antigen forlane 1, polypeptide SDS-PAGE size standards; lane 2, MGN-1191; lane 3,MGN-1191/pYA3167, transformant #1 (χ8281); lane 4, MGN-1191/pYA3167,transformant #2; lane 5, MGN-1191/pYA3167, transformant #3; lane 6,MGN-1256; lane 7, MGN-1256/pYA3167, transformant #1 (χ8280); lane 8,MGN-1256/pYA3167, transformant #2; lane 9, MGN-1256/pYA3167,transformant #3; and lane 10, χ6212/pYA3167.

FIG. 16 illustrates the pCMV beta-asd plasmid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based upon the discovery made in S.typhimurium, which is predictive for other Salmonella such as S. typhi,that Salmonella having a functional ropS gene and an RpoS⁺ phenotypehave a high immunogenicity and can be advantageously used as vaccinesand as carrier microbes. Such vaccines and carrier microbes can serve asvehicles for delivering desired gene products such as antigens to humansas well as for delivering nucleic acids, either DNA or RNA, to targethuman cells.

The ropS gene product contributes to the virulence of Salmonellatyphimurium in mice, at least in part, by regulating expression ofchromosomal gene determinants of virulence and is believed to contributeto S. typhi virulence in humans through a similar mechanism. Much of thework that has led to the development of live S. typhi vaccines forimmunization of humans has relied upon studies using strains of S.typhimurium tested in mice. These S. typhimurium strains cause aninvasive infection in susceptible mice that resembles typhoid in humans.(Carter and Collins, J. Exp. Med. 139:1189-1203; Hohmann et al., InfectImmun 22:763-770, 1978; Coynaut et al. Molecular Microbiol. 22:149-160,1996). Furthermore, the role of the rpoS gene in the invasiveness andvirulence of Salmonella typhimurium is relevant to the invasiveness andvirulence of Salmonella typhi which lack a virulence plasmid inasmuch asstrains of Salmonella typhimurium cured of the virulence plasmid havebeen shown to colonize Peyers patches with efficiency similar to that ofthe wild-type microorganisms (Gulig and Curtiss, Infect Immun55:2891-2901, 1987; Hackett et al., J Infect Dis 153:1119-1125, 1986).The results of studies in Salmonella typhimurium, which are thus alsoapplicable to Salmonella typhi, show that the rpoS gene product controlsthe expression of chromosomally encoded genes which are important forinvasiveness and virulence. (Nickerson and Curtiss, Infect and Immun65:1814-1823, 1997; Kowarz et al, J Bacteriol 176:6852-6860, 1994).

In studies described in the Examples below, the inventors herein foundthat the presence of a functional rpoS gene is necessary for the earlystages of the Salmonella typhimurium infection process at the level ofthe Peyer's patches and that the rpoS gene product acts through aninteraction with chromosomal genes. In particular, it was discoveredthat an rpoS mutant of S. typhimurium exhibited wild-type abilities toattach to and invade cells of a human embryonic intestinal epithelialcell line, Int-407, and a murine macrophage-like cell line, J774. Inaddition, mutation in the rpoS gene did not affect the intracellularsurvival of S. typhimurium in either the J774 macrophage-like cells orrat bone marrow-derived macrophages. However, the rpoS mutantdemonstrated a decreased ability to colonize murine Peyer's patchesafter oral inoculation as compared to its wild-type virulent parentstrain.

In addition, virulence plasmid-cured derivatives of the rpoS mutant wererecovered in lower numbers from murine Peyer's patches than wereplasmid-cured derivatives of the isogenic wild-type S. typhimurium. Thisindicates that RpoS regulation of chromosomally-encoded genes isimportant for colonization of the murine gut associated lymphoid tissue(GALT) by S. typhimurium.

Microscopic analysis of histological sections taken from Peyer's patchesafter peroral infection of mice showed that, unlike its wild-typevirulent parent strain, the isogenic ropS mutant did not destroy thefollicle-associated epithelium of the GALT. Furthermore, the ropS mutantdemonstrated a decreased ability to adhere to histological sections ofmurine Peyer's patches as compared to its wild-type parent. These dataimplicate the rpoS gene in the initial stages of systemic infection bySalmonella involving interaction of Salmonella with cells of the Peyer'spatches.

As a result of the decreased ability of rpoS mutants to colonize Peyer'spatches, earlier reports have suggested that Salmonella strains havingan inactivating mutation in the rpoS gene are attractive candidates foruse in live oral attenuated vaccines. (Nickerson and Curtiss, supra,1996). In contrast to this earlier work, however, the present inventionis directed to Salmonella strains and other bacteria having a functionalrpoS⁺ gene along with an attenuating mutation in another gene. As aresult, the strains of the present invention are able to colonizePeyer's patches, or similar tissues including, for example, otherlymphoid tissues of the GALT in humans, without destroying the invadedcells in order to achieve a high immunogenicity upon administrationorally. Furthermore, the M cells of the follicle-associated lymphoidtissue of the GALT are functionally, morphologically and structurallythe same as the M cells associated with other mucosal associatedlymphoid tissues (MALT) in the body such as conjunctiva associatedlymphoid tissue (CALT), bronchus associated lymphoid tissue (BALT) andnasal associated lymphoid tissue (NALT), as well as lymphoid tissues inthe rectum, etc. Thus, it is believed that the presence of a functionalrpoS⁺ gene in the Salmonella will play an important role in the invasionand colonization of these tissues when administration is by routesincluding oral, intranasal, rectal, etc. In fact, as shown in theexamples below, RpoS⁺ S. typhimurium, both non-recombinant andrecombinant expressing a foreign antigen, are superior to isogenic RpoS⁻S. typhimurium strains in conferring protective immunity and ineliciting antibody responses to the foreign antigens when deliveredintranasally where colonization of the NALT and BALT should be of primeimportance.

The Salmonella and other bacterial strains within the scope of thepresent invention can be selected on the basis of their having afunctional rpoS⁺ gene which produces a functional rpoS gene product. TherpoS gene product is known to be a stationary-phase sigma factor whichis responsible for the control of a regulon of over 30 genes expressedin response to starvation, during the transition to stationary phase andin response to stresses. Protein products of genes under the control ofRpoS regulate a number of cell functions including protection againstDNA damage, the determination of morphological changes, the mediation ofvirulence, osmoprotection, thermotolerance (Loewen and Hengge-Aronis,Annu. Rev. Microbiol. 48:53-80, 1994) and acid tolerance (Lee et al.,Mol. Microbiol. 17:155-167, 1995). Many of these stresses areencountered by bacteria during infection or immunization of an animal orhuman host. Reference to RpoS phenotype herein is intended to mean themanifestation of cell functions regulated by ropS gene expression in themicrobe.

Many of the cell functions controlled by RpoS regulation can be assessedin determining the RpoS phenotype of a microbe. For example, one cananalyze cultures for catalase production. This test is based upon RpoSpositive regulation of the katE gene, which produces hydroperoxidase IIcatalase. The culture medium of strains carrying the wild-type rpoSallele grown to stationary phase, bubble vigorously upon addition ofhydrogen peroxide, whereas minimal bubbling occurs in the stationaryphase culture medium of strains carrying a mutant rpoS allele (Lowen, J.Bacteriol. 157:622-626, 1984; Mulvey et al., Gene 73:337-345, 1988). TheRpoS phenotypes of the attenuated S. typhimurium strains can also beassayed by determining the sensitivity of these strains to nutrientdeprivation, acid or oxidative stresses, and defective glycogenbiosynthesis ability. In a variation of this approach, the RpoSphenotype could be determined by P22HTint-mediated transduction of theropS allele into wild-type S. typhimurium χ3339, with subsequent testingof the derived microbe for catalase production as described above.

One can also genetically alter a strain which does not contain afunctional rpoS⁺ gene using conjugation, transformation, or transductionto introduce a functional recombinant rpoS⁺ gene which provides an RpoS⁺phenotype in the catalase test. The recombinant rpoS⁺ gene can be fromany suitable homologous or heterologous source, preferably a homologoussource.

It may also be possible to introduce into Salmonella containing afunctional rpoS⁺ gene another functional recombinant rpoS⁺ gene on aplasmid replicon or integrated into the chromosome to further enhancethe expression of genes regulated by the RpoS protein. This might bedesirable in certain situations such as, for example, in microbes havingdiminished rpoS gene expression, i.e., microbes which display nonoptimalcolonization of the GALT, or even in microbes in which the rpoS geneexpression is not diminished but a greater than normal expression isdesired.

It is also possible to provide a Salmonella or other bacteria strainthat is able to effectively colonize the GALT or other lymphoid tissueseven though it does not express functional RpoS. For example, the RpoS⁻phenotype could be circumvented by incorporating into an rpoS mutantstrain at least one recombinant virulence gene. Recombinant virulencegene or recombinant RpoS virulence gene as referenced herein is intendedto mean that the recombinant gene is capable of expressing a geneproduct having the same biological function, i.e. facilitating effectivecolonization of the GALT or other lymphoid tissue, as that of achromosomal virulence gene normally regulated by RpoS. However,expression of the incorporated recombinant virulence gene is controlledby regulatory elements that are not dependent upon the presence offunctional RpoS, thereby providing expression of the recombinantvirulence gene product in the absence of functional RpoS. For example, afunctional rpoS⁺ gene is shown to be important for adherence bySalmonella to Peyer's patches, which is necessary for colonization ofthis tissue. One or more genes responsible for this adherence isbelieved to be regulated by RpoS. One group of candidate genescontrolling adherence to Peyer's patches that may be regulated by RpoSmay be the lpf fimbrial operon (Bäumler et al., Proc. Natl. Acad. Sci.,USA 93:279-283, 1996). Thus, the invasiveness and immunogenicity of anrpoS mutant microbe can be enhanced by transforming the microbe with oneor more virulence genes under the control of regulatory elements thatare not dependent upon the presence of functional RpoS.

In one embodiment of the present invention, the rpoS⁺ bacterial strains,in particular rpoS⁺ Salmonella strains, are attenuated derivatives of apathogenic strain. By derivative or derived strain reference is made toa strain that has been genetically modified from its parent from whichit is descended. By pathogenic it is meant that the microbe is capableof causing disease or impairing normal physiological functioning.Reference to avirulence or attenuation herein, is intended to mean thata particular microbe strain is incapable of inducing a full suite ofsymptoms of the disease state that is normally associated with itsvirulent non-attenuated pathogenic counterpart. Thus, avirulence orattenuation includes a state of diminished virulence or ability toproduce disease conditions and the attenuated or avirulentmicroorganisms are not necessarily completely absent of any ability toimpair normal physiological functioning of the host. In addition, anattenuated or avirulent microbe is not necessarily incapable of everfunctioning as a pathogen, but the particular microbe being used isattenuated with respect to the particular individual being treated.

The rpoS⁺ strains of the present invention, including rpoS⁺ Salmonellastrains, are attenuated by virtue of their containing an attenuatingmutation in one or more genes that renders the microorganism attenuated.In a preferred embodiment, the strains have at least two mutations eachof which act to attenuate the microorganism and which, in combination,significantly increase the probability that the microorganism will notrevert to wild-type virulence. Mutations can be insertions, partial orcomplete deletions or the like so long as expression of the gene isdiminished and virulence is decreased. Attenuating mutations can be inbiosynthetic genes, regulatory genes and/or genes involved in virulence.(See Doggett and Brown, supra). Examples of mutations include, but arenot limited to a mutation in a pab gene, a pur gene, an aro gene, asd, adap gene, nada, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya,crp, dam, phoP, phoQ, rfc, poxA, galU, metL, metH, mviA, sodC, recA,ssrA, ssrB, sirA, sirB, sirC, inv, hilA, hilC, hilD, rpoE, flgM, tonB,slyA, and combinations thereof. The skilled artisan will readilyappreciate that any suitable gene mutation can be used in the presentinvention so long as the mutation of that gene renders the microorganismattenuated.

Methods are known in the art that can be used to generate mutations toproduce the attenuated microbes of the present invention. For example,the transposon, Tn10, can be used to produce chromosomal deletions in awide variety of bacteria, including Salmonella (Kleckner et al., J. Mol.Biol. 116:125-159, 1977; EPO Pub. No. 315,682; U.S. Pat. No. 5,387,744).

Recently, new methods have become available for producing specificdeletions in genes. These methods involve initially selecting a gene inwhich the deletion is to be generated. In one approach the gene can beselected from a genomic library obtained commercially or constructedusing methods well known in the art (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Press, ColdSpring Harbor, N.Y.). Clones containing the gene are isolated from thegenomic library by complementation of a strain which contains a mutationin the same gene. Alternatively, when the DNA sequence of the gene isknown, selected primers for the polymerase chain reaction method (PCR)can amplify the gene, often with some flanking sequence, from a sampleof bacteria or from purified genomic DNA and the PCR product can beinserted into a cloning vector.

A specific deletion in the selected gene can be generated by either oftwo general methods. The first method generates a mutation in a geneisolated from a population of clones contained in a genomic DNA libraryusing restriction enzymes and the second method generates the mutationin a gene of known sequence using PCR.

Using the first method, the position of the gene on a vector isidentified using transposon tagging and a restriction map of therecombinant DNA in the vector is generated. Information derived from thetransposon tagging allows all or a portion of a gene to be excised fromthe vector using the known restriction enzyme sites.

The second method which is based upon PCR methodology can be used whenthe DNA sequence of the gene is known. According to this method,divergent PCR primers amplify the upstream and downstream regionsflanking a specified segment of DNA to be deleted from the gene andgenerate a PCR product consisting of the cloning vector and upstream anddownstream flanking nucleotide sequences (Innes et al. Eds., PCRProtocols, 1990, Academic Press, New York). In a variation of thismethod, PCR products are produced representing portions of the gene orflanking sequence, which are then joined together in a cloning vector.

The DNA containing the mutant gene can be introduced into the bacterialhost by transformation using chemical means or electroporation, byrecombinant phage infection, or by conjugation. In preferred embodimentsthe mutant gene is introduced into the chromosomes of the bacteria whichcan be accomplished using any of a number of methods well known in theart such as, for example, methods using temperature-sensitive replicons(Hamilton et al., J. Bacteriol. 171:4617-4622, 1989), lineartransformation of recBC mutants (Jasin et al., J. Bacteriol.159:783-786, 1984), or host restricted replicons known as suicidevectors (Miller et al., J. Bacteriol. 170:2575-2583, 1988). Theparticular method used is coupled with an appropriate counter selectionmethod such as, for example, fusaric acid resistance or sucroseresistance followed by subsequent screening for clones containing themutant allele based upon phenotypic characteristics or by using PCR,nucleic acid hybridization, or an immunological method.

The attenuated rpoS⁺ bacteria strains of the present invention, inparticular, attenuated S. typhi mutants, can be used in the form ofvaccines to deliver recombinant antigens to a human or nucleic acids totarget cells of a human. Thus, it is apparent that the present inventionhas wide applicability to the development of effective recombinantvaccines against bacterial, fungal, parasite or viral disease agents inwhich local immunity is important and might be a first line of defense.Some examples are recombinant vaccines for the control of bubonic plaguecaused by Yersinia pestis, of gonorrhea caused by Neisseria gonorrhoea,of syphilis caused by Treponema pallidum, and of venereal diseases aswell as eye infections caused by Chlamydia trachomatis or of pneumoniacaused by C. pneumoniae. Species of Streptococcus from both group A andgroup B, such as those species that cause sore throat or heart disease,Neisseria meningitidis, Mycoplasma pneumoniae, Haemophilus influenzae,Bordetella pertussis, Mycobacterium tuberculosis, Mycobacterium leprae,Streptococcus pneumoniae, Brucella abortus, Vibrio cholerae, Shigellaspecies, Legionella pneumophila, Helicobacter pylori, Campylobacterjejuni, Borrelia burgdorferi, Rickettsia species, Pseudomonasaeruginosa, and pathogenic E. coli such as ETEC, EPEC, UTEC, EHEC, andEIEC strains are additional examples of microbes within the scope ofthis invention from which genes could be obtained. Recombinantanti-viral vaccines, such as those produced against influenza viruses,are also encompassed by this invention. Recombinant anti-viral vaccinescan also be produced against viruses, including RNA viruses such asPicornaviridae, Caliciviridae, Togaviridae, Flaviviridae, Coronaviridae,Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae,Bunyaviridae, Arenaviridae, Reoviridae or Retroviridae; or DNA virusessuch as Hepadnaviridae, Paroviridae, Papovaviridae, Adenoviridae,Herpesviridae or Poxviridae. Recombinant vaccines to protect againstinfection by pathogenic fungi, protozoa or parasites are alsocontemplated by this invention. All of the above examples of pathogensare provided for illustrative puropSes and are not intended to beconstrued in a limiting sense.

Thus, in one set of embodiments, the present invention can be describedas a vaccine for the immunization of a human comprising a liveattenuated derivative of a pathogenic bacteria such as a pathogenic S.typhi wherein the derivative contains a functional rpoS gene andexpresses an RpoS⁺ phenotype. The attenuated bacteria is also capable ofexpressing a recombinant gene derived from an organism that is apathogen of or that produces an allergen of the human.

In embodiments in which the immunogenic component of the vaccine is anallergen of the host, such a vaccine can be used in an exposure regimendesigned to specifically desensitize an allergic host. Allergies topollens, mold spores, insect parts, animal dander and the like are dueto the inhalation of air and/or ingestion of feed containing suchallergens. The allergies that result are associated with a presence ofIgE antibodies that bind to allergens which activate mast cells forrelease of histamines. As is well known, desensitization againstallergens can be achieved by repetitive parenteral immunization ofextracts containing the allergen. Likewise, it is known that oralingestion of raw honey containing pollens can be used to effectivelyinduce a state of tolerance against those allergens. Oral ingestion withsuch allergens can on the one hand induce an SIgA response that couldblock the ability of allergens to react with IgE and mast cells or ifadministered in sufficient quantity could serve to suppress thesynthesis of IgE antibodies, that is to induce tolerance. Since thespecific allergenic molecule in many allergens has been identified andthe cDNA cloned to obtain the nucleotide sequence specifying theallergen, it is now possible to genetically engineer heterologous hostcells to express the allergen (see for example, Valenta et al, Allergy53:552-561. 1998; Olsson et al., Clin. Exp. Allergy 28:984-991. 1998;Soldatova et al., J. Allergy Clin. Immunol. 101:691-698, 1998; Asturiaset al, Clin Exp Allergy 27:1307-1313; Twardosz et al, Biochem BiophysRes Commun 239:197-204, 1997). Accordingly, the attenuated RpoS⁺Salmonella of the present invention can be engineered to express anallergen, possibly in a modified immunogenic but nonallergenic form toinduce a state of tolerance or to actively promote the production ofSIgA against the allergen. The RpoS⁺ attenuated Salmonella describedherein have been shown to be effective in eliciting immune responsesand, hence, it follows that use of such RpoS⁺ Salmonella to expressmodified allergens would be likely to be effective in ameliorating theconsequences of exposure of humans to allergens by inhalation oringestion.

In other embodiments, the recombinant gene expresses a gamete-specificantigen which is capable of eliciting an immune response that confers anantifertility effect upon the immunized individual (See, U.S. Pat. No.5,656,488).

The attenuated microbes of this invention can additionally be used asvectors for the synthesis of various host proteins. Because theattenuated microbes of this invention are able to traverse a variety ofimmunocompetent structures including gut-associated lymphoid tissue(GALT), mesenteric lymph nodes and spleen after introduction into thehost, such microbes can be used to target a variety of immunoregulatoryproducts. Accordingly, one or more genes encoding immunoregulatoryproteins or peptides can be recombinantly introduced into the attenuatedmicrobes such that when the microbes taking up residence in theappropriate immunocompetent tissue are capable of expressing therecombinant product to suppress, augment or modify the immune responsein the host. Examples of immunoregulatory molecules include but are notlimited to: colony stimulating factors (macrophage, granulocyte, ormixed), macrophage chemotoxin, macrophage inhibition factor, leukocyteinhibitory factors, lymphotoxins, blastogenic factor, interferon,interleukins, tumor necrotizing factor, cytokines, and lymphokines.

The attenuated microbes of the present invention are also contemplatedfor use to deliver and produce pharmacologically active products thatmight stimulate or suppress various physiological functions (i.e.,growth rate, blood pressure, development of sexual maturity etc.). Insuch microbes, the recombinant gene encodes said pharmacologicallyactive products.

The recombinant gene of the microbes of the present invention can beincorporated into a “balanced-lethal” system which selects formicroorganisms containing and capable of expressing the recombinant geneby linking the survival of the microorganism to the continued presenceof the recombinant gene. “Balanced-lethal” mutants of this type arecharacterized by a lack of a functioning native chromosomal geneencoding an enzyme which is essential for cell survival, preferably anenzyme which catalyzes a step in the biosynthesis of diaminopimelic acid(DAP) and even more preferably a gene encoding beta aspartatesemialdehyde dehydrogenase (Asd). DAP pathway enzymes and Asd arerequired for cell wall synthesis. The mutants also contain a firstrecombinant gene which can serve to complement the non-functioningchromosomal gene and this is structurally linked to a second recombinantgene encoding the desired product. Loss of the complementing recombinantgene causes the cells to die by lysis when the cells are in anenvironment where DAP is lacking. This strategy is especially usefulsince DAP is not synthesized by eukaryotes and, therefore, is notpresent in immunized host tissues. Methods of preparing these types of“balanced lethal” microbes are disclosed in U.S. Pat. No. 5,672,345.

By immunogenic agent is meant an agent used to stimulate the immunesystem of an individual, so that one or more functions of the immunesystem are increased and directed towards the immunogenic agent.Immunogenic agents include vaccines. Immunogenic agents can be used inthe production of antibodies, both isolated polyclonal antibodies andmonoclonal antibodies, using techniques known in the art.

An antigen or immunogen is intended to mean a molecule containing one ormore epitopes that can stimulate a host immune system to make asecretory, humoral and/or cellular immune response specific to thatantigen.

An epitope can be a site on an antigen to which an antibody specific tothat site binds. An epitope could comprise 3 amino acids in a spatialconformation which is unique to the epitope; generally, an epitopeconsists of at least 5 amino acids and more usually, at least 8-10 aminoacids. The term “epitope” is intended to be interchangeable with theterm “antigenic determinant” as used herein. The term “epitope” is alsointended to include T-helper cell epitopes in which an antigenicdeterminant is recognized by T-helper cells through association withmajor histocompatibility complex class II molecules. In addition, theterm epitope includes any antigen, epitope or antigenic determinantwhich is recognized by cytotoxic T cells when presented by a MHC class Imolecule on the surface of an antigen presenting cell. A cytotoxic Tcell epitope can comprise an amino acid sequence of between about 6 toabout 11 amino acids, and preferably comprises a sequence of 8 or 9amino acids.

By vaccine is meant an agent used to stimulate the immune system of anindividual so that protection is provided against an antigen notrecognized as a self-antigen by the immune system. Immunization refersto the process of inducing a continuing high level of antibody and/orcellular immune response in which T-lymphocytes can either kill thepathogen and/or activate other cells (e.g., phagocytes) to do so in anindividual, which is directed against a pathogen or antigen to which theorganism has been previously exposed. Although the phrase “immunesystem” can encompass responses of unicellular organisms to the presenceof foreign bodies, in this application the phrase is intended to referto the anatomical features and mechanisms by which an individualproduces antibodies against an antigenic material which invades thecells of the individual or the extra-cellular fluid of the individualand is also intended to include cellular immune responses. In the caseof antibody production, the antibody so produced can belong to any ofthe immunological classes, such as immunoglobulins, A, D, E, G or M. Ofparticular interest are vaccines which stimulate production ofimmunoglobulin A (IgA) since this is the principle immunoglobulinproduced by the secretory system of warm-blooded animals, althoughvaccines of the invention are not limited to those which stimulate IgAproduction. For example, vaccines of the nature described herein arelikely to produce a broad range of other immune responses in addition toIgA formation, for example cellular and humoral immunity. Immuneresponses to antigens are well studied and widely reported. A survey ofimmunology is provided in Elgert, Klaus D., Immunology, Wiley Liss,Inc., (1996); Stites et al., Basic & Clinical Immunology; 7th Ed.,Appleton & Lange, (1991) the entirety of which are incorporated hereinby reference.

An “individual” treated with a vaccine of the present invention isdefined herein as referring to a human host.

Microbes as used herein can include bacteria, protozoa and bothunicellular and multicellular fungi. The term parasite as used herein isintended to include protozoans such as species of Plasmodium andToxoplasma as well as species of Entamoeba, Leishmania and Trypanosomaand helminths such as trematodes, cestodes and nematodes. Viruses asused herein can include RNA viruses such as, for example,Picornaviridae, Caliciviridae, Togaviridae, Flaviviridae, Coronaviridae,Rhabdoviridae, Filoviridae, Paramyxoviridae,Orthomyxoviridae,Bunyaviridae, Arenaviridae, Reoviridae and Retroviridae; and DNA virusessuch, for example, as Hepadnaviridae, Paroviridae, Papovaviridae,Adenoviridae, Herpesviridae and Poxviridae.

Reference to a recombinant gene is intended to mean genetic materialthat is transferred by human intervention from a first organism into asecond organism which upon reproduction gives rise to descendantscontaining the same genetic material. Generally, such exchange ofgenetic material from the first organism to the second organism eitherdoes not take place or rarely takes place in nature.

The term gene as used herein in its broadest sense represents anybiological unit of heredity. It is not, however, necessary that therecombinant gene be a complete gene as is present in the parent organismand capable of producing or regulating the production of a macromoleculesuch as for example, a functioning polypeptide. The recombinant genemay, thus, encode all or part of an antigenic product. Furthermore, therecombinant gene can also include DNA sequences that serve as promoters,enhancers or terminators and DNA sequences that encode repressors oractivators that regulate expression of a recombinant gene encoding allor part of an antigen. A recombinant gene can also refer to gene fusionswhich encode polypeptide fusion products. The encoded gene product can,thus, be one that was not found in that exact form in the parentorganism. For example, a functional gene coding for a polypeptideantigen comprising 100 amino acid residues can be transferred in partinto a carrier microbe so that a peptide comprising only 75, or even 10,amino acid residues is produced by the cellular mechanisms of the hostcell. However, if this gene product can serve as an antigen to causeformation of antibodies against a similar antigen present in the parentorganism or as a T-cell epitope recognized by T-helper cells, the geneis considered to be within the scope of the term gene as defined in thepresent invention. Alternatively, if the amino acid sequence of aparticular antigen or fragment thereof is known, it is possible tochemically synthesize the DNA fragment or analog thereof by means ofautomated gene synthesizers or the like and introduce said DNA sequenceinto the appropriate expression vector. This might be desirable in orderto use codons that are preferred codons for high level expression inSalmonella. At the other end of the spectrum is a long section of DNAcoding for several gene products, one or all of which can be antigenic.For example, such a long section of DNA could encode 5 to 15 proteinsnecessary for the synthesis of fimbrial antigens (fimbriae), whichmediate adhesion of pathogens to host cells (Bäumler et al., supra). Theinduction of an immune response against fimbriae can provide protectionagainst the pathogen. Thus, a gene as defined and claimed herein is anyunit of heredity capable of producing an antigen. The gene can be ofchromosomal, plasmid, or viral origin. It is to be understood that theterm gene as used herein further includes DNA molecules lacking intronssuch as, for example, is the case for CDNA molecules, so long as the DNAsequence encodes the desired gene product. Further, the term geneincludes within its meaning RNA molecules that serve as genes of RNAviruses or the complement of such RNA molecules wherein the RNA moleculeor complement thereof can serve as an mRNA to be transcribed into aviral protein which is immunogenic. The term gene as used herein alsoincludes a DNA sequence specifying the viral strand that serves as anmRNA to be translated into the viral protein which is immunogenic.

In order for the gene to be effective in eliciting an immune response,the gene must be expressed. Expression of a gene means that theinformation inherent in the structure of the gene (the sequence of DNAbases) is transformed into a physical product in the form of an RNAmolecule, polypeptide or other biological molecule by the biochemicalmechanisms of the cell in which the gene is located. The biologicalmolecule so produced is referenced as the gene product. The term geneproduct as used here refers to any biological product or productsproduced as a result of the biochemical reactions that occur under thecontrol of a gene. The gene product can be, for example, an RNAmolecule, a peptide, or a product produced under the control of anenzyme or other molecule that is the initial product of the gene, i.e.,a metabolic product. For example, a gene can first control the synthesisof an RNA molecule which is translated by the action of ribosomes intoan enzyme which controls the formation of glycans in the environmentexternal to the original cell in which the gene was found. The RNAmolecule, the enzyme, and the glycan are all gene products as the termis used here. Any of these as well as many other types of gene products,such as glycoproteins, glycolipids and polysaccharides, will act asantigens if introduced into the immune system of an individual. Proteingene products, including glycoproteins and lipoproteins, are preferredgene products for use as antigens in vaccines.

In order for a vaccine to be effective in stimulating cellular immunityor in producing antibodies, the antigenic materials must be releasedand/or presented in such a way to trigger the induction of a cellularimmunity and/or induce the antibody-producing mechanism of thevaccinated individual. Therefore, the microbe carrier of the geneproduct must be introduced into the individual. In order to stimulate apreferred response of the gut-associated lymphoid tissue (GALT) orbronchus-associated lymphoid tissue (BALT), introduction of the microbeor gene product directly into the gut or bronchus is preferred, such asby oral administration, gastric intubation or intranasally in the formof aerosols, although other methods of administering the vaccine, suchas intravenous, intramuscular,. subcutaneous injection or intramammaryor intrapenial or vaginal or rectal administration, are possible.

The attenuated microbe can be used as a carrier microbe, for example,for an antigen or for a DNA or RNA vaccine vector, and once the carriermicrobe is present in the individual, the antigen needs to becomeavailable to the individual's immune system. In the case of a carriermicrobe for delivery of a nucleic acid molecule, the nucleic acidmolecule needs to be released within the target cell. This can beaccomplished when the carrier microbe dies so that the antigen moleculesor nucleic acid molecules are released. Of course, the use of “leaky”attenuated mutants that release the contents of the periplasm withoutlysis is also possible.

Alternatively, a gene can be selected that controls the production of anantigen that will be made available by the carrier cell to the outsideenvironment prior to the death of the cell. In this way, it is possibleto use a viable microbe that will persist in the vaccinated individual,for example in its Peyer's patches or other GALT, NALT or BALT, etc.,and continue to produce antigen, thereby continually inducing antibodyformation and/or a cellular immune response. A preferred gene productunder these circumstances is a product that is transferred through thecell membrane of the attenuated carrier microbe into the externalenvironment or a product that becomes attached to or embedded in theexternal membrane so that all or part of the gene product is exposed tothe environment. Typical of this latter type of gene product areantigens normally found on the surface of the organism against whichprotection is desired. If these antigens are transported to thebacterial cell surface in a normal manner, antibody formation againstthe antigens will be enhanced.

Nucleic acid vaccines are well known in the art (see e.g., Ulmer et al.,Amer. Soc. Microbiol. News 62:476-479, 1996; Ulmer et al., Curr.Opinion. Immunol. 8:531-536, 1996; and Robinson, H. L., Vaccine15:785-787, 1997) and delivery of DNA vaccines by attenuated bacteriawith subsequent stimulation of an immune response against theprotein-encoded by the DNA vaccine has been described (Sizemore et al.,Vaccine 15:804-806, 1997). Thus, it is expected that the attenuatedmicrobes of the present invention can also be used as delivery vehiclesfor DNA vaccines. Typically, bacteria containing such DNA vaccines donot themselves express the gene product encoded by the DNA vaccine, butrelease the DNA vaccine into one or more human tissues, where the geneproduct is then expressed by host cell transcription and translationmachinery. However, it is also contemplated that a DNA vaccine forimmunization against RNA viruses can be constructed in which copies ofthe RNA viral genome, or of a protein-encoding portion thereof, will bemade in the cytoplasm of the attenuated bacteria. Such RNA moleculeswould be released into the human tissues, e.g., by lysis of theattenuated bacteria, where they would serve as mRNA for synthesis ofimmunogenic viral protein(s). It is also contemplated that the DNAvaccine vector within the attenuated bacterial host could synthesize themRNA for a desired gene product within the bacteria which could then bedelivered to the eukaryotic cell where the mRNA would be directlytranslated into the desired gene product. In this case, the mRNA wouldpossess information or signals that caused translation to be dependenton the eukaryotic host cell and which would preclude, for the most part,translation within the attenuated bacterial cell.

The use of pathogens to deliver antigens from other pathogens to theGALT or BALT would be inappropriate if it were not for the fact thatsuch pathogens can be rendered attenuated while retaining ability tocolonize these tissues.

The organism from which the recombinant gene is derived can be any humanpathogen or may be an organism that produces an allergen or otherantigen to which a human can be sensitive. Allergens are substances thatcause allergic reaction, in this case in the human which will bevaccinated against them. Many different materials can be allergens, suchas animal dander and pollen, and the allergic reaction of individualswill vary for any particular allergen. It is possible to inducetolerance to an allergen in an individual that normally shows anallergic response. The methods of inducing tolerance are well-known andgenerally comprise administering the allergen to the individual inincreasing dosages. Further discussion of tolerance induction is givenin the Barrett textbook previously cited. Lastly, the host individualitself can serve as a source of genetic material when immunoregulatorygenes or genes for other pharmacologically active substances are beingexpressed by the vectors.

Administration of a live vaccine of the type disclosed above to anindividual can be by any known or standard technique. These include oralingestion, gastric intubation, or broncho-nasal-ocular spraying. All ofthese methods allow the live vaccine to easily reach the NALT, GALT orBALT cells and induce antibody formation and cell mediated immunity andare the preferred methods of administration. Other methods ofadministration, such as intravenous injection, that allow the carriermicrobe to reach the individual's blood stream can be acceptable.Intravenous, intramuscular or intramammary injection are also acceptablewith other embodiments of the invention, as is described later.

Any of a number of commonly used recombinant DNA techniques can be usedin producing the attenuated microbes of the present invention which arecapable of expressing a recombinant gene. Following ligation to aplasmid, phage or cosmid vector the recombinant molecules so formed canbe transferred into a host cell by various means such as conjugation, ortransformation (uptake of naked DNA from the external environment, whichcan be artificially induced by the presence of various chemical agents,such as calcium ions), including electroporation. Other methods such astransduction are also suitable, wherein the recombinant DNA is packagedwithin a phage such as transducing phage or cosmid vectors. Once therecombinant DNA is in the carrier cell, it may continue to exist as aseparate autonomous replicon or it may insert into the host cellchromosome and be reproduced along with the chromosome during celldivision.

Once the genetic material has been transferred, the microbes containingthe transferred genetic material are selected.

The immunization dosages required will vary with the antigenicity of thegene product and need only be an amount sufficient to induce an immuneresponse. Routine experimentation will easily establish the requiredamount. Multiple dosages are used as needed to provide the desired levelof protection.

The pharmaceutical carrier or excipient in which the vaccine issuspended or dissolved may be any solvent or solid or encapsulatingmaterial such as for a lypholized form of the vaccine. The carrier isnon-toxic to the inoculated individual and compatible with themicroorganism or antigenic gene product. Suitable pharmaceuticalcarriers are known in the art and, for example, include liquid carriers,such as normal saline and other non-toxic salts at or near physiologicalconcentrations, and solid carriers, such as talc or sucrose. Gelatincapsules can serve as carriers for lypholized vaccines. Adjuvants may beadded to enhance the antigenicity if desired. When used foradministering via the bronchial tubes, the vaccine is preferablypresented in the form of an aerosol. Suitable pharmaceutical carriersand adjuvants and the preparation of dosage forms are described in, forexample, Remington's Pharmaceutical Sciences, 17th Edition, (Gennaro,Ed., Mack Publishing Co., Easton, Pa., 1985).

Immunization of an individual with a pathogen-derived gene product canalso be used in conjunction with prior immunization with the attenuatedderivative of a pathogenic microorganism acting as a carrier to expressthe gene product specified by a recombinant gene from a pathogen. Suchparenteral immunization can serve as a booster to enhance expression ofthe secretory immune response once the secretory immune system to thatpathogen-derived gene product has been primed by immunization with thecarrier microbe expressing the pathogen-derived gene product tostimulate the lymphoid cells of the GALT or BALT. The enhanced responseis known as a secondary, booster, or anamnestic response and results inprolonged immune protection of the host. Booster immunizations may berepeated numerous times with beneficial results.

In another embodiment, the present invention provides a method forassessing the immunogenicity of a Salmonella comprising determining theRpoS phenotype of the Salmonella. The method is also applicable forother bacteria such as Shigella and E.coli and Salmonella-Shigellahybrids, Salmonella-E.coli hybrids and Shigella-E.coli hybrids. Thepresence of an RpoS⁺ phenotype confers upon the microbe the ability toinvade and colonize the lymphoid tissue associated with the particularroute of administration used such as, for example, the GALT followingoral administration or other lymphoid tissues, such as the NALT or BALTfollowing other routes of administration. This in turn results in a highlevel of immunogenicity. Thus, detecting the presence of an RpoS⁺phenotype indicates that the microbe will have a high level ofimmunogenicity compared to a microbe that is RpoS⁻, but otherwisegenetically identical.

The RpoS⁺ phenotype can be assessed by determining the properties of themicrobe. This can be done by any of a number of possible methods. Forexample, by analyzing cultures for catalase production. This test isbased upon RpoS positive regulation of the katE gene, which produceshydroperoxidase II catalase. The culture medium of strains carrying thewild-type rpoS allele bubble vigorously upon addition of hydrogenperoxide, whereas minimal bubbling occurs in the culture medium ofstrains carrying a mutant rpoS allele (Lowen, J. Bacteriol. 157:622-626,1984; Mulvey et al., Gene 73:337-345, 1988). Other methods can also beused for determining the RpoS phenotypes of the attenuated Salmonella orother bacteria strains including determining the sensitivity of thestrains to nutrient deprivation, acid or oxidative stresses, anddefective glycogen biosynthesis ability. In a variation of thisapproach, the rpoS allele can be transduced into a wild-type Salmonellaand the resultant derivative Salmonella tested for RpoS phenotype.

One can also assess the RpoS⁺ phenotype by determining the genetic makeup of the microbe wherein the presence of a functional rpoS⁺ genecapable of producing a functional rpoS gene product indicates an RpoS⁺phenotype.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

GENERAL METHODS

The bacterial strains used in the present studies were constructed usingthe following general materials and methods. Listings of phages,plasmids and micro-organisms used in constructing the strains are givenin Tables 1 and 2.

TABLE 1 Microorganisms Strain Relevant Designation GenotypeSource/Reference/Derivation Salmonella typhi Strains χ3743 ISP1804 Type46 1983 isolate from Chilean patient, received from D. Hone, Center forVaccine Development, MD χ3744 ISP1820 Type 46 cys trp ATCC 55116; 1983isolate from Chilean patient; received from D. Hone χ3745 ISP2822 TypeE1 ATCC 55114; 1983 isolate from Chilean patient; received from D. Honeχ3746 ISP2825 Type E1 1983 isolate from Chilean patient; received fromD. Hone χ3769 Ty2 Type E1 rpoS cys Louis Baron, Walter Reed ArmyInstitute of Research χ3927 Ty2 Δcrp-11 Δ[zhb::Tn10] ATCC 55117 Δcya-12Δ[zid-62::Tn10] χ4073 Ty2 Δ[crp-cdt]-10 ATCC 55118 Δ[zhb::Tn10] Δcya-12Δ[zid-62::Tn10] χ8203 cys trp ATCC 9992V; AMC strain Boxhill 58V χ8204cys trp ATCC 33458; CDC 2862-79 χ8205 Ty21a galE rpoS cys trp ATCC33459; CDC 2861-79 χ8206 cys trp aroA serC ATCC 39926; Stanford 531Ty;derivative of CDC10-80 purA155 χ8207 cys trp ATCC 10749; AMC 42-A-63χ8208 Ty2 cys ATCC 19430; NCTC 8385 χ8209 cys trp ATCC 9993; AMC 42-A-63MGN-1018 Ty2 rpoS cys ΔphoPQ23 MEGAN Health, Inc., St. Louis, MOMGN-1038 ISP1820 cys trp ΔphoPQ23 MEGAN Health, Inc., St. Louis, MOMGN-1191 ISP1820 cys trp ΔphoPQ23 MEGAN Health, Inc., St. Louis, MOΔasdA16 MGN-1256 Ty2 rpoS cys ΔphoPQ23 MEGAN Health, Inc., St. Louis, MOΔasdA16 χ8280 MGN-1256(pYA3167) χ8281 MGN-1191(pYA3167) χ8434 Ty2 rpoS⁺cys ΔphoPQ23 MGN-1013 by introducing recombinant rpoS⁺ gene from pYA3467χ8435 Ty2 rpoS+ cys ΔphoPQ23 χ8280 by introducing recombinant rpoS⁺ genefrom ΔasdA16 (pYA3167) pYA3467 χ8438 Ty2 Type E1 rpoS⁺ cys χ3769 byintroducing recombinant rpoS⁺ gene from (RpoS⁺) pYA3467 Salmonellatyphimurium Strains χ3000 LT2-Z prototroph Received from C. Turnboughχ3181 SR-11 pStSR100⁺ wild Isolated by passage from murine Peyer'spatch. type Gulig and Curtiss, Infect. Immun. 65:2891-2901 (1987) χ3339SL1344 pStSL100⁺ hisG Animal passaged isolate of SL1344, isolated fromrpsL, colicin⁺ liver of moribund mouse after p.o. infection. Gulig andCurtiss, Infect. Immun. 65:2891-2901 (1987) χ3340 SL1344 pStSL100⁻ hisGVirulence plasmid-cured derivative of χ3339; Gulig rpsL, colicin⁺ andCurtiss, Infect. Immun. 65:2891-2901 (1987). χ3420 SL1344 hisG rpsl xy1P22HTint(χ3376)→χ3339 with selection for Tc^(R) Mot⁻ fli-8007::Tn10Fla⁻. χ3422 SR-11 fli-8007::Tn10 P22HTint(χ3376)→χ3181 with selectionfor Tc^(R) Mot⁻ Fla⁻ χ3679 SR-11 ΔaroA554 P22HTint(χ3678)→χ3181selecting Tc^(r) and screening for Aro⁻ followed by selection fortetracycline sensitivity, Aro⁻. χ3761 UK-1 wild-type ATCC 68169;spleenic isolate from infected chick. prototroph χ4937 UK-1 rpoS::RR10P22HTint(SF1005)→χ3761 with selection for ampicillin resistance χ4973SL1344 pStSL100⁺ hisG Nickerson and Curtiss, Infect. Immun.,65:1814-1823 rpsL rpoS::RR10, (1997) colicin⁺ χ8125 SL1344 pStSL100⁻hisG P22HTint(SF1005)→χ3340 with selection for ampicillin rpsLrpoS::RR10, resistance; Nickerson and Curtiss, Infect. Immun., colicin⁺65:1814-1823 (1997) χ8214 UK-1 rpcS::RR10 Δcya-27P22HTint(SF1005)→MGN-431 with selection for Δcrp-27 ampicillinresistance χ8215 SR-11 rpoS::RR10 P22HTint(SF1005)→χ3679 with selectionfor ampicillin ΔaroA554 resistance χ8217 UK-1 rpoS::RR10 Δcya-27P22HTint(SF1005)→MGN-232 with selection for ampicillin resistance χ8296SL1344 pStSL100⁺ Δcya-28 MEGAN Health, Inc.; χ3339 derivative with threeΔcrp-27 ΔasdA16 (RpoS⁺) defined deletion mutations χ8309 SL1344pStSL100t Δcya-28 P22HTint(χ4973)→χ8296 Δcrp-27 ΔasdA16 rpoS (RpoS⁻)MGN-232 UK-1 Δcya-27 MEGAN Health, Inc.; defined cya deletion derivativeof χ3761 MGN-431 UK-1 Δcya-27 Δcrp-27 MEGAN Health, Inc.; defined crpdeletion derivative of MGN-232 ATCC 14028s prototroph, Tet^(s) wild-typeinvasive strain obtained from E. Heffron SF1005 14028s rpoS::RR10 F.Fang, Univ. Colorado Health Sci. Center Shigella flezneri 2a Strains3457T wild-type Curtiss collection 15D Δasd::kan Sizemore et al.(Science, 270:299-302, 1995; Vaccine 15:804-807, (1997) E. coli Strainsχ6101 K-12 DH5α F⁻ Ø80d lacZ Guy Cardineau, Sungene Technologies, Inc.,San Jose, ΔM15 Δ(lacZYA-argF)-4169 CA gln44 λ⁻ gyrA recA1 relA1 endA1hsdR17 (r_(k)− ,m_(k)+) χ6212 K-12 F⁻ Ø80d lacZ ΔM15 This labΔ(lacZYA-argF)-4169 supE44 λ⁻ gyrA recA1 relA1 endA1 ΔasdA4Δ[zhf-2::Tn10] hsdR17 (r_(k)−,m_(k)+) MGN-026 K-12 F⁻ Ø80d lacZ ΔM15λpir→χ6101; MEGAN Health, Inc., St. Louis, MO Δ(lacZYA-argF)-4169 supE44λpir gyrA recA1 relA1 endA1 hsdR17 (r_(k)−,m_(k)+) MGN-617 thi-1 thr-1leuB6 supE44 MEGAN Health, Inc., St. Louis, MO tonA21 lacY1 recA RP4-2-Tc::Mu λpir, ΔasdA4 Δ[zhf-2::Tn10]

TABLE 2 Phages and Plasmids Bacteriophage Description Source/ReferenceP22HTint high frequency Schmeiger, Mol. Gen. Genet. 119:75-88, 1972;Jackson generalized transducing et al., J. Mol. Biol. 154:551-563, 1982;Ray et al., mutant of the temperate Mol. Gen. Genet. 135:175-184, 1974.lambdoid phage P22 P22 H5 clear plaque forming Casjens et al., J. Mol.Biol. 194:411-422, 1987. mutant of P22HTint Plasmids pSK::rpoS S.typhimurium 14028 F. Fang, Univ. Colorado Health Sci. Center rpoS⁺ genecloned into the EcoRV site of pBlueScript/SK pMEG-003 pir-dependent R6Kori MEGAN Health, Inc., St. Louis, MO Tc^(r) asd⁺ pMEG-006 pir-dependentR6K ori Megan Health, Inc., St. Louis, MO Tc^(r) ΔasdA16 pMEG-068Contains phoQ gene MEGAN Health, Inc., St. Louis, MO pMEG-149 Amp^(R)mobilizable pir- MEGAN Health, Inc., St. Louis, MO dependent suicidevector; containing the sacBR genes from B. subtillis, RK2 mob, R6K oripMEG-210 phoQ deletion of pMEG- MEGAN Health, Inc., St. Louis, MO 068pMEG-213 Derivative of pMEG-149 MEGAN Health, Inc., St. Louis, MOcontaining phoPQ23 defined deletion of pMEG-210 pMEG-328 Derivative ofpNEB-193 MEGAN Health, Inc., St. Louis, MO containing the S. typhimuriumUK-1 rpoS⁺ gene cloned into the Bam HI and XbaI sites pMEG-375 cat genefrom pACYC184 MEGAN Health, Inc., St. Louis, MO cloned into PMEG-149pNEB-193 pUC19 derivative that New England Biolabs carries singlerestriction sites for unique 8bp cutters AscI, PacI and PneI within thepolylinker region pYA3167 asd - complementing Nardelli-Haefliger et al.,Infect. Immun. 64:5219- plasmid; expresses the 5224, 1996 Hepatitis Bvirus (HBV) nucleocapsid pre-S1 and pre-S2 epitopes on HBV core pYA3342Asd⁺ cloning vector This lab with pBR replicon pYA3433 contains rpoS⁺gene This lab cloned from pSK::rpoS into SmaI site of pMEG- 149 pYA3467contains S. typhimurium This lab UK-1 rpoS⁺ gene from pMEG-328 clonedinto PmeI and Sma I sites of pMEG-375

Bacterial strains were maintained as duplicate −70° C. frozen culturessuspended in 1% Bacto-peptone (Difco) containing 5% glycerol and werealso stored at −20° C. in 1% Bacto-peptone and 50% glycerol for routineuse. Bacteria were generally cultured in L Broth (Lennox, Virology1:190-206, 1965) or Luria Broth (Luria et al., J. Bacteriol. 74:461-476,1957). Agar plates contained 1.5% Difco Agar. Carbohydrate utilizationwas evaluated by supplementing MacConkey (Difco) or Eosin Methylene Blueagar base (Curtiss, Genetics 58:9-54, 1968) with 1% final concentrationof an appropriate carbohydrate. Minimal liquid (ML) and minimal agar(MA) were prepared as described in Curtiss (J. Bacteriol. 89:28-40,1965) and supplemented with nutrients at optimal levels. Buffered salinewith gelatin (BSG) was. used routinely as a diluent (Curtiss, 1965supra).

Bacteriophage P22HTint was used for transduction using standard methods(Davis et al., A Manual for Genetic Engineering—Advanced BacterialGenetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1979). An overnight culture of a donor strain was diluted 1:20 intoprewarmed Luria broth, grown for one hour with shaking at 37° C., andthen infected with P22HTint at a multiplicity of infection (MOI) of0.01. The infection mixture was shaken overnight or for approximatelyfifteen hours. A few drops of chloroform were added to ensure completebacterial cell lysis, and the mixture was allowed to shake an additionalten minutes at 37° C., then centrifuged at 7,000 rpm in a Sorvall SS-34rotor for ten minutes to remove bacterial debris. The supernatant fluidwas extracted and removed to a clean tube with a drop or two of freshchloroform and stored at 4° C. This method generally provides a phagelysate containing about 10¹⁰ PFU/ml titered on χ3000. Tetracycline wasused in plates at 12.5 μg/ml to select for Tn10 transductants,Tn10-induced mutations, or merodiploid strains expressing theTn10-derived tetracycline-resistance genes from a chromosomallyintegrated suicide vector. The Tn10 transposon excises from thechromosome at a low frequency, often deleting a portion of the genomeflanking the transposon. Cells which undergo an excision event alsobecome sensitive to tetracycline, and can be identified by plating onmedia containing fusaric acid, which kills tetracycline-resistantbacteria (Maloy and Nunn, J. Bacteriol. 145:1110-1112, 1981).Tetracycline-sensitive strains which have lost an integrated suicideplasmid along with the plasmid linked tetracycline-resistance genes canalso be selected on fusaric acid media.

Tetracycline-resistant cultures were grown standing overnight in L brothcontaining 12.5 μg/ml tetracycline at 37° C. to approximately 5×10⁸CFU/ml. These cultures were diluted 1:40 into prewarmed L broth withouttetracycline and aerated at 37° C. to a titer of about 2×10⁹ CFU/ml,serially diluted into BSG, and plated from these dilutions onto fusaricacid media. Fusaric acid resistant colonies were selected afterincubation for 48 hours at 37° C. Fusaric acid resistant isolates wererestreaked to fusaric acid media, then patched to Penassay agar (Difco)with and without tetracycline to confirm the loss of the Tn10-derivedantibiotic resistance element. Other phenotypes were scored whereindicated using appropriate media.

Suicide vectors containing an ampicillin-resistance gene, asucrose-utilization cassette, and an incP mobilization site wereconstructed. Mutant genes which have been introduced into these plasmidscan be introduced into the bacterial chromosome after transformation, orpreferably by conjugation, to generate ampicillin-resistant (100 μg/ml)merodiploids. Such merodiploids can be grown on media containing 5%sucrose to select for the loss of the integrated plasmid along with theampicillin-resistance and sucrose-utilization genes.Ampicillin-sensitive strains can be phenotypically characterized for thepresence of appropriate defined deletion mutant alleles.

Improved selection of merodiploids can be achieved by introducing a catgene conferring resistance to chloramphenical (40 μg/ml) in addition toan ampicillin-resistance gene on the suicide vector. After merodiploidformation, selection on media with 5% succrose leads to loss of bothdrug-resistance genes and the succrose-utilization genes. Theserecombinants can then be screened for the desired allele replacement.

EXAMPLE 1

This example illustrates the role of the rpoS gene in efficient invasionand colonization of the GALT by S. typhimurium using an rpoS mutantstrain, χ4973, compared to its wild-type parent, χ3339.

Strain Construction

χ3339 is a wild-type, virulent, animal-passaged isolate of S.typhimurium strain SL1344 described in Gulig et al. (Infect Immun55:2891-2901, 1987). SF1005 is an rpoS::RR10 mutant derived from S.typhimurium strain ATCC 14028s and containing an ampicillin resistancegene linked to the rpoS::RR10 mutant allele (Fang et al., Proc. Nat'l.Acad. Sci., USA 89:11978-11982, 1992). The mutant rpoS::RR10 allele wasmoved into χ3339 using a P22HTint transducing phage lysate prepared onSF1005 and selecting for ampicillin resistance (Ap^(r)) due to thepresence of the β-lactamase gene (bla) linked to the RR10 insertion inthe ropS gene. The allelic exchange between SF1005 and χ3339 wasconfirmed by Southern blot analysis, and the resulting χ3339 rpoS::RR10mutant derivative was designated as χ4973. Transductants were screenedfor sensitivity to P22HTint by cross streaking with P22H5, a clearplaque mutant. Pseudolysogenic colonies were distinguished fromnon-lysogens on Evans blue and uranine (EBU) indicator agar (Sternberget al., Meth. Enzymol. 204:2-43, 1991). Media were supplemented with 50μg ampicillin per ml when required to select for χ4973.

The presence of smooth lipopolysaccharide (LPS) in χ4973 was confirmedusing the method of Hitchcock et al. (J. Bacteriol. 154:269-277, 1983).LPS was silver stained by the method of Tsai et al. (Anal Biochem119:115-119, 1982). This experiment showed that the mutation in rpoS didnot affect LPS structure.

Virulence of an RpoS Mutant in Mice

The virulence of χ3339 was compared to that of the rpoS mutant strainχ4973 upon oral inoculation of eight- to ten-week old female BALB/cmice. Animal inoculation for the determination of the fifty per centlethal dose (LD₅₀) was performed as described earlier with minormodifications (Gulig et al., Infect Immun 55:2891-2901, 1987). Mice weredeprived of food and water for four to six hours prior to peroralinoculation. Gastric acidity was not neutralized prior to infection.LD₅₀ titers were determined according to the method of Reed and Muench(Am. J. Hyg. 27:493-497, 1938) for each strain using results obtainedfrom four mice per inoculum dose evaluated for a period of thirty days.

The peroral LD₅₀ for the ropS mutant strain χ4973 was greater than 8×10⁹colony forming units per dose. This value represented more than a fourlog increase over the oral lethal dose of 8×10⁵ colony forming unitsobserved for the wild-type parent strain χ3339. This result isconsistent with Fang et al., supra, who reported a three log increase inthe oral LD₅₀ dose for SF1005, as compared to the rpoS⁺ parent strainATCC 14028s. Further studies were then conducted to determine why therpoS mutant strain was attenuated compared to its wild-type parent.

Comparative Testing of Attachment, Invasion and Survival

Human embryonic intestinal epithelial cell line Int-407 (Henle et al.,J. Immunol. 79:54-59, 1957) and murine macrophage-like cell line J774(Ralph et al., Nature 257:393-394, 1975) were used to examine the effectof rpoS on the adherence and invasive abilities of S. typhimurium. Eachcell line was maintained in Minimal Essential Medium (MEM; GibcoBRL,Grand Island, N.Y.) containing Hank's Balance Salt Solution (HBSS;GibcoBRL), 2 mM glutamine, and 10% fetal calf serum (FCS; HyClone,Logan, Utah) at 37° C. in an atmosphere containing 5% Co₂. Cells werepassaged every two to three days with medium changes. Macrophagemonolayers used in an infection assay were prepared by gently scrapingpassaged cells into solution, diluting the cell suspension, inoculatingwells of a 24-well microtiter dish, and incubating at 37° C. in a 5% CO₂environment. Int-407 cells were distributed in a similar fashion, butwere trypsinized for removal from monolayers.

Bacterial attachment and invasion assays using cells from the humanintestinal epithelial cell line, Int-407, and the mouse macrophage-likecell line, J774, followed methods according to Galan et al., Proc NatlAcad Sci, USA 86:6383-6387, 1989, with minor modifications. Bacteriawere grown as static cultures in L broth at 37° C. to mid log phase orabout 0.5 optical density as measured at 600 nm. Because expression ofrpoS and RpoS-regulated genes increases as cells enter into stationaryphase, a control culture was also grown statically for four days tosaturation in order to establish the maximal level of rpoS expression.Bacterial cultures were washed and resuspended in HBSS immediately priorto infection of monolayers. Int-407 monolayer attachment and invasionwas allowed to proceed for two hours at 37° C. in MEM in an atmosphereof 5% CO₂ and at an MOI of between two and ten bacterial cells perInt-407 cell. Attachment and invasion assays using J774 cells wereperformed as with Int-407 cells, except that only one hour was allowedfor adherence and invasion. As a control for distinguishing adhesionfrom phagocytosis of bacterial cells by the monolayer cells, J774 cellswere monitored at 4° C. in the presence of bacteria according to themethod of Lee et al., Proc. Nat'l . Acad. Sci., USA 87:4304-4308, 1990.

Infected monolayers were washed three times with isotonicphosphate-buffered saline (PBS) after the attachment and invasionincubation, and then lysed with PBS containing 0.1% sodium deoxycholateto assess the total number of bacteria associated with the culturedcells. Duplicate monolayers infected in parallel were incubated anadditional two hours with MEM containing 10 μg/ml gentamicin in order tokill extracellular bacteria prior to lysis so that the number ofinternalized bacteria could be enumerated. Viable bacterial cell countswere obtained by plating dilutions of lysed monolayers onto L agar andincubating at 37° C. for eighteen to twenty four hours. Results areshown in Tables 3 and 4 below.

TABLE 3 Effect of an rpoS::RR10 mutation on adherence to and invasion ofInt-407 cells by S. typhimurium χ3339 and its rpoS mutant derivative,χ4973^(a) Growth Percent Percent Strain phase adhesion^(b) invasion^(c)χ3339 Exponential 59.2 ± 0.3 83.0 ± 26.9 χ4973 Exponential 51.4 ± 0.288.0 ± 22.0 χ3339 Stationary 12.8 ± 2.3 25.0 ± 2.4  χ4973 Stationary34.0 ± 18.0 50.0 ± 4.0  ^(a)The data are given as the means ± SEM forthree trials. ^(b)Percent of inoculum adherent to cells after incubationfor 2 hours. ^(c)Percent of inoculum recovered after incubation for 2additional hours in gentamicin [10 μg/ml].

TABLE 4 Effect of an rpoS::RR10 mutation on adherence to and invasion ofJ774 cells by S. typhimurium χ3339 and its rpoS mutant derivative,χ4973^(a) Growth Percent Percent Strain phase adhesion^(b) invasion^(c)χ3339 Exponential 55 ± 4.1 66 ± 3.7 χ4973 Exponential 57 ± 1.5 46 ± 4.4χ3339 Stationary 44 ± 3.2 14 ± 3.5 χ4973 Stationary 19 ± 2.4 11 ± 0.3^(a)The data are given as the means ± SEM for three trials. ^(b)Percentof inoculum adherent to cells after incubation for 1 hour. ^(c)Percentof inoculum recovered after incubation for 2 additional hours ingentamicin [10 μg/ml].

When the S. typhimurium strains were grown to exponential phase, therpoS::RR10 mutant, χ4973, attached to Int-407 and J774 cells to the sameextent as its wild-type parent, χ3339 (Tables 3 and 4). Percent invasionwas also the same for both strains in the intestinal epithelial cellline, Int-407 (Table 3). However, invasion showed a small decrease withthe rpoS::RR10 mutant in the macrophage cell line, J774, compared to thewild-type parent (Table 4). These data indicate that when the S.typhimurium were in the exponential growth phase, the rpoS genecontributed little or nothing to the ability of the bacteria to attachto or invade into the cells. When the bacterial cells were in thestationary phase, however, results were equivocal. Whereas adhesion andinvasion were slightly increased with rpoS::RR10 mutants grown tostationary phase in Int-407 cells, adhesion was slightly decreased forthe rpoS::RR10 mutants grown to stationary phase in J774 cells (Tables 3and 4, respectively). In additional studies not shown, no difference wasobserved in the ability of these strains to adhere to or invade intoJ774 cells when assays were conducted at 4° C. (data not shown). Thesedata indicate that the ropS gene product has little or no effect on invitro attachment to or invasion of intestinal epithelial cells andmacrophage-like cells during the exponential and stationary growthphases of S. typhimurium and are consistent with what has been reportedfor SL1344-derived S. typhimurium containing an altered ropS allele fromS. typhimurium LT-2 (Wilmes-Riesenberg et al., Infec. Immun. 65:203-210,1997).

S. typhimurium bacteria having an ropS mutation were also able tosurvive when internalized in J774 murine macrophage-like cells or ratbone-marrow derived macrophages. Rat bone-marrow derived macrophageswere obtained from the femurs and tibias of Sprague Dawley rats (HarlanSprague Dawley, Indianapolis, Ind.) and grown in a 75 cm² flaskcontaining Dulbecco Minimal Essential Medium (DMEM; GibcoBRL, GrandIsland, N.Y.) containing 10% fetal calf serum (FCS), 100 unitspenicillin/ml and 100 μg streptomycin/ml for 10 days. The macrophageswere then cultured in DMEM containing 10% fetal calf serum (FCS), 5%horse serum (HS; Sigma, St. Louis, Mo.), 10% L-cell-conditioned medium,1 mM glutamine, and 1% penicillin for twenty four hours at 37° C. in anenvironment containing 5% CO₂. Nonadherent cells were removed, spentmedium was replaced, and the cells were incubated an additional fivedays. Macrophages were gently scraped from the surface of the flask,resuspended in fresh DMEM supplemented with 10% FCS, 5% HS and 10%L-cell conditioned medium without antibiotics and used to seed wells ofa 24-well microtiter plate prior to infection experiments, at aconcentration of 5×10⁵ or 1×10⁶ cells per ml of rat bone marrow-derivedmacrophages or J774 cells, respectively.

χ3339 or χ4973 were grown to stationary phase as described above andused in an intracellular survival assay in J774 cells or rat bone-marrowderived macrophages according to Buchmeier et al., Infect. Immun.57:1-7, 1989, with minor modifications. Bacteria were opsonized with 10%normal mouse serum for thirty minutes prior to infection of themonolayers prepared above at a multiplicity of infection (MOI) ofbetween two and ten bacteria per cell. Infected monolayers wereincubated for twenty minutes to allow for invasion or phagocytosis, andthen washed two times with PBS to remove bacteria remaining in solutionphase. Fresh growth media containing 10 μg/ml gentamicin was added towashed, infected monolayers to eliminate extracellular bacterial growth.Infected monolayers were incubated for the indicated times aftergentamicin addition, washed to remove traces of antibiotic, and thenlysed with 0.1% sodium deoxycholate in PBS. Dilutions of lysates wereplated onto L agar and incubated at 37° C. for twenty four to thirty sixhours in order to enumerate surviving intracellular bacteria.

FIGS. 1 and 2 illustrate the log of the mean and standard deviations ofcounts of bacteria associated with cells obtained from three wells overthe time course of 24 hours. Both χ3339 and χ4973 exhibited a decreasein bacterial cell count during the first two to four hours, followed byan increase in cell count during the next 20 hours in J774 cells.However, a decrease in the viable number of these bacteria was observedbetween 4 and 20 hours in rat bone marrow macrophages, yet significantnumbers of bacteria survived during the course of the study with littledifference between the rpoS wild-type or rpoS mutant.strains. Thus, S.typhimurium rpoS mutants are able to survive in either murinemacrophage-like J774 cells or in rat bone marrow-derived macrophages aswell as their wild-type parent, indicating that the rpoS gene productplays little or no role in the survival of the microbe in thesemacrophages.

Tissue Distribution of rpoS Mutants after P.O. Infection

To compare the GALT colonization abilities of the rpoS::RR10 mutant andwild-type strains, animal infectivity studies were performed.

Bacteria used in these studies were grown aerobically in a volume of 100ml L broth at 37° C. to an optical density of 0.8 as measured at 600 nm.Bacteria were harvested by centrifugation for ten minutes at 7,000 rpm.The cell pellet was resuspended in 1 ml buffered saline with gelatin(BSG).

Eight- to ten-week old female BALB/c mice purchased from Charles RiverLaboratories (Wilmington, Mass.) were either coinfected with both theχ4973 rpoS mutant and χ3339 wild-type strains or individually infectedwith each strain. In each of the coinfection and individual infectionexperiments, four groups of three mice each were perorally inoculatedwith approximately equal numbers of bacteria. Mice were euthanized byCO₂ asphyxiation at one hour and at one, three and five days after oralinoculation. Organs and tissues of interest were aseptically removed andhomogenized with a tissue homogenizer (Brinkman Instruments). Five toten lymphoid follicles representing the Peyer's patches were collectedfrom each mouse and combined before homogenization. Homogenates werediluted into BSG and plated onto MacConkey/1% lactose agar with andwithout ampicillin. This allows a comparison between the total number ofboth wild-type and ropS mutant Salmonella typhimurium which successfullycolonize the tissues, to the total number of rpoS mutant bacteria whichsuccessfully colonize the same tissues. The data for the coinfection andindividual infection experiments are shown below in Tables 5 and 6,respectively.

TABLE 5 Ratios of S. typhimurium wild-type to rpoS mutants in mousetissues after peroral coinfection^(a) Time after Intestinal IntestinalPeyer's infection Contents Wall^(b) Patches Spleen Liver 1 hour 1.7 ±0.4 1.7 ± 0.5 N.D.^(c) N.D.^(c) N.D.^(c) 1 day 2.1 ± 0.4 1.5 ± 0.1  1.1± 0.10 N.D.^(c) N.D.^(c) 3 days 2.1 ± 0.1 8.7 ± 4.3 10.9 ± 5.4  815 ±743 122 ± 38  5 days 2.7 ± 0.2 6.0 ± 4.2 469 ± 325 250,000 ± 11,750 ±249,800 10,250 ^(a)Approximately equal numbers of χ3339 (wild-type) andχ4973 rpoS (4.5 × 10⁹ and 4.0 × 10⁹ colony forming units (CFU),respectively) were administered perorally to 10-week old BALB/c mice.Mean ratios of CFU/g of tissue for χ3339/χ4973 ± SEM (n = 3) are given.Only bacterial counts greater than 20 CFU/g were considered whencalculating the ratios. ^(b)Small and large intestine with Peyer'spatches removed. ^(c)N.D., bacterial numbers were not determined.

TABLE 6 Colonization of mouse tissues after individual infection with S.typhimurium wild-type or rpoS mutant strains^(a) Bacterial numbers(cfu/g tissue) Time^(b) Tissue χ3339 χ4973 Day 3 Wall^(c) 2.1 × 10³ ±1.2 × 10³ 2.7 × 10³ ± 6.4 × 10² Peyer's 1.7 × 10⁵ ± 4.1 × 10⁴ 5.8 × 10⁴± 1.1 × 10⁴ patches Day 5 Wall^(c) 1.9 × 10⁴ ± 6.6 × 10³ 6.5 × 10³ ± 2.5× 10³ Peyer's 9.9 × 10⁵ ± 2.4 × 10⁵ 4.5 × 10⁴ ± 1.6 × 10⁴ patches^(a)Ten-week old BALB/c mice were administered perorally with eitherwild-type χ3339 (2.7 × 10⁹ CFU) or rpos mutant χ4973 (1.1 × 10⁹ CFU)bacteria. Only bacterial counts greater than 20 CFU/g were consideredsignificant. ^(b)The intestinal wall and the Peyer's patches wereexcised after the indicated time. Three mice were euthanized at eachtime point. Standard errors are shown for each experiment. ^(c)Small andlarge intestine with Peyer's patches removed.

The rpoS mutant strain χ4973 and the wild-type strain χ3339 initiallycolonized the gastrointestinal tract with similar efficiency as judgedby the numbers of bacteria associated with the intestinal wall at daythree in both mixed (Table 5) and individual (Table 6) infections. Thusthe ropS mutants survived passage through the stomach as well as thewild-type parent strain.

However, the rpoS mutant strain χ4973 was much less efficient incolonizing the Peyer's patches as compared to its wild-type parentstrain, χ3339 (Tables 5 and 6). This disadvantage of the ropS strain waseven more pronounced in the spleen (Table 5). Thus, the S. typhimuriumstrain with the rpoS mutant allele is defective in its ability tocolonize the GALT and the spleen, which are two primary lymphoid organsin which immune responses are elicited.

To determine whether the ropS gene product regulates expression ofchromosomally-encoded genes whose products are important for S.typhimurium colonization of Peyer's patches, the wild-type χ3339 andrpoS mutant χ4973 strains were cured of their virulence plasmids togenerate plasmid-cured isogenic derivatives χ3340 and χ8125,respectively. The ability of these derivative strains to colonizePeyer's patches was examined following peroral administration of χ3340and χ8125 in a 1:1 ratio and the data are shown in Table 7 below.

TABLE 7 Ratios of wild-type to rpoS mutants for virulence plasmid-curedS. typhimurium in mouse tissues after peroral coinfection^(a) Time afterIntestinal Intestinal Peyer's Infection Contents Wall^(b) Patches 3 days37.7 ± 11.8 4.4 ± 3.5 UD^(c) 5 days 3.2 ± 1.2 1.2 ± 0.3 5.4 ± 0.5^(a)Approximately equal numbers of χ3340 and χ8125 (4.0 × 10⁹ CFU and3.4 x 10⁹ CFU, respectively) were administered perorally to 10-week oldBALB/c mice. Mean ratios of CFU/g of tissue for χ3340/χ8125 ± SEM (n =3) are given. Only bacterial counts greater than 20 CFU/g wereconsidered when calculating the ratios. ^(b)Small and large intestinewith Peyer's patches removed. ^(c)Bacterial numbers undetectable at a1:100 dilution.

As shown in Table 7, χ8125, the RpoS⁻ derivative of χ3340, which in turnis the virulence plasmid-cured derivative of the SL1344 wild-typestrain, χ3339, exhibited a reduced ability (ca. 5.4 fold) to colonizePeyer's patches at 5 days postinfection as compared to the colonizingability of χ3340. These data indicate that RpoS regulates expression ofchromosomally-encoded gene(s) whose products are important forsuccessful colonization of murine Peyer's patches after oralinoculation.

Effect of RpoS⁻ Strain on Histology of Peyer's Patches

Peyer's patches were removed from the intestinal wall of mice at varioustimes after peroral inoculation with χ3339 or χ4973 and were immediatelyfixed in an ice-cold solution of 1.5% glutaraldehyde and 1.5%paraformaldehyde in a 0.1M sodium phosphate buffer, pH 7.4, followed byfixation in 2.5% glutaraldehyde also in sodium phosphate buffer, pH 7.4for one hour at room temperature. Thick sliced sections of fixed tissuewere stained with Epoxy Tissue Stain (Electron Microscopy Sciences, FortWashington, Pa.) to locate domes of the Peyer's patches. Thin slicedsections were examined with a Hitachi H-600 transmission electronmicroscope (TEM) operated at 75 kV accelerating voltage.

Observation of sections using light or TEM microscopy revealed majormorphological changes in the integrity of the Peyer's patch epitheliumas early as one day after oral inoculation with the wild-type virulentstrain χ3339 (FIGS. 3E and 3F). The destruction of thefollicle-associated epithelium (FAE) at three and five days after oralinoculation with χ3339 was even more apparent as seen in FIGS. 3G and3H. The enterocytes had been completely sloughed from the domeepithelium and extensive tissue necrosis was observed. In addition,there was a dramatic decrease in cell density of the Peyer's patchlymphoid follicle tissue five days after oral inoculation of mice withχ3339 (FIGS. 3h and 4 c).

In contrast, Peyer's patches from mice that were uninfected or infectedwith the rpoS mutant strain χ4973 did not exhibit the dramatic changesin tissue morphology caused by χ3339 infection. Instead, the integrityof the dome epithelium was uncompromised and very little decrease incell density of the underlying lymphoid tissue was observed at one,three and five days after oral inoculation (FIGS. 3B-3D).

TEM analysis of Peyer's patch tissue before and five days after oralinoculation with the rpoS mutant χ4973 showed that the FAE remainedintact (FIGS. 5A and 5B), whereas the FAE was totally destroyed in χ3339infected Peyer's patches as early as one day after oral inoculation(FIG. 5F).

Dramatic morphological changes in the underlying lymphoid tissue werealso clearly apparent when viewed by TEM. Five days after infection withχ4973, lymphoid cells within the Peyer's patch follicle appeared healthyand similar in morphology to Peyer's patches from uninfected mice (FIGS.5A and 5B). In contrast, extensive changes in gross morphology wereobserved in Peyer's patch lymphoid cells five days after infection withχ3339 (FIG. 5C).

These data show that rpoS mutant S. typhimurium do not efficientlyinvade and colonize the GALT. As a result, the ropS mutants would beexpected to be defective in stimulating a generalized mucosal immuneresponse, which is dependent upon colonization of the GALT. Furthermore,because the GALT is the portal of entry into mesenteric lymph nodes andthe spleen, the mutants would also be expected to be ineffective ininvading and colonizing these deeper lymphoid tissues. This would beexpected to result in the mutants being defective in stimulatingsystemic humoral immunity as well as cellular immune responses, whichare dependent upon colonization of the mesenteric lymph nodes andspleen. In contrast, strains containing the wild-type rpoS allele moreefficiently invade and colonize the GALT and deeper lymphoid tissues andare, as a result, more effective in eliciting mucosal, hurmoral andcellular immune responses.

On the other hand, the rpoS⁺ strains destroyed the Peyer's patch tissue,making such strains less than ideal candidates for use in vaccines.Therefore, it is desirable to modify the rpoS⁺ microbes with at leastone virulence reducing mutation so that the microbes are still able toinvade and colonize the Peyer's patches, but without destroying thePeyer's patch tissue.

EXAMPLE 2

This example illustrates methods which can be used in constructingdefined deletion mutations in genes to confer an attuation upon rpoS⁺ S.typhimurium and S. typhi strains as well as other bacteria suitable foruse as vaccines for humans.

The generation of chromosomal deletions using transposon Tn10 has beenpreviously described in a wide variety of bacteria, including Salmonella(Kleckner et al., J. Mol. Biol. 116:125-159, 1977; EPO Pub. No. 315,682;U.S. Pat. No. 5,387,744; which are incorporated by reference). Recently,new methods have become available for introducing specific mutationsinto genes. The gene to be mutated can be selected from a population ofclones contained in a genomic DNA library constructed in a cloningvector, or by cloning the amplified product containing all or a portionof the gene into a plasmid using PCR methodology. Mutations introducedinto such genes or portions of genes are known as defined deletions andthese are constructed using one of two general methods.

One method employs restriction enzymes to remove all or a portion of anisolated gene from a recombinant vector. This method allows the mutationof genes for which DNA sequence information is unavailable. However,this method is limited to the use of restriction sites present withinthe gene or within the DNA flanking the cloned gene.

Another method employs the use of divergent PCR primers synthesizedbased upon known DNA sequence either within the gene to be deleted orwithin DNA flanking the gene. The primers are mixed with a vectorcontaining a cloned gene and subjected to an inverse PCR reaction,resulting in the amplification of the entire plasmid but deleting all ora portion of the target gene (Innis et al., infra). The PCR reactionamplifies upstream and downstream regions flanking a specified segmentof DNA to be deleted from the cloned gene and generates a productconsisting of the cloning vector and upstream and downstream flankingsequences. The inverse PCR method is preferred because it allows theplacement of mutations of any size at any position within a gene ofknown DNA sequence, and allows the introduction of novel restrictionsites to be engineered into the PCR primers or target DNA which then canbe used for the subsequent insertion of other cloned sequences. Analternative PCR method for generating defined deletions relies onamplified PCR products which represent portions of the gene or flankingDNA sequence. These are ligated together in a cloning vector toconstruct the defined deletion mutation.

A genomic library can be constructed in any number of cloning vectors(Sambrook et al., supra). Clones containing a gene in which a deletionis to be generated can be isolated from the genomic library bycomplementation in a bacterial strain which contains a mutation in thesame gene.

For example, genomic DNA libraries from wild-type Salmonella typhimuriumUK-1 (χ3761) can be constructed in a suitable cloning vector such aspNEB-193 (New England Biolabs), which is a pUC19 derivative that carriessingle sites for the unique 8-base cutters: AscI, PacI and PmeI.Generally, genomic DNA is isolated according to standard methods(Sambrook et al., Molecular Cloning/A Laboratory Manual Second Edition,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989). Sau3A1partially digested genomic DNA is sized on an agarose gel and extractedusing commercially available methods in kit form obtained from Promega,Quiagen, or Bio101. DNA fragments between 2 and 6 kb are isolated andligated into a plasmid first digested with BamHI or BglII, thendephosphorylated using alkaline phosphatase according to themanufacturers' instructions. The resulting plasmid library is thenintroduced into an appropriate E. coli strain in order to amplify thegenomic library and to obtain a population of recombinant plasmidscontaining random genomic DNA inserts ranging in size from 2 to 6 kb.Relevant clones are isolated from a genomic library by complementationof mutant E. coli or S. typhimurium strains.

Where the DNA sequence of a gene is already known, PCR primers aresynthesized and the gene and often some flanking sequence is amplifiedusing PCR methodology directly from a sample of bacteria or frompurified genomic DNA, and the product, cloned into a plasmid vector suchas pNEB-193. Thus, where the gene sequence is known, screening a genomiclibrary is not required.

Virtually any cloning vector can be used in constructing the strains ofthe present invention, so long as the defined deletion is located on thevector and is linked to a selectable marker. There are a number ofdifferent methods available for introducing the defined deletionmutations into the chromosome, including temperature-sensitive replicons(Hamilton et al., J. Bacteriol. 171:4617-4622, 1989), lineartransformation of recBC mutants (Jasin et al., J. Bacteriol.159:783-786, 1984), and host restricted replicons known also as suicidevectors (Miller et al., J. Bacteriol. 170:2575-2583, 1988). All of thesemethods can result in an allele replacement, whereby a mutant alleleconstructed on a vector replaces a wild-type allele on the chromosome,or vice versa.

The pir-dependent R6K replicon has been used by numerous investigatorsand is one of the most reliable suicide vectors available for allelereplacement. Replication of the R6K plasmid requires the pir geneproduct. A pir-dependent plasmid will not replicate in a pir⁻ hostbacterium, and so the presence of a defined deletion mutation on apir-dependent plasmid will allow for the selection of rare events inwhich the plasmid has integrated into the host chromosome within ahomologous region flanking the deletion constructed on the plasmid. Thisevent will confer some selectable phenotype upon the strain into whichthe plasmid has integrated, because even though the plasmid cannotreplicate, the integration event provides a mechanism of stablemaintenance of the elements on the plasmid. Antibiotic-resistanceelements are generally used to select for the presence of the integratedplasmid, and can be selected from genes which encode resistance toampicillin, kanamycin, chloramphenicol, gentamicin, spectinmycin andtetracycline, and others well known in the art. The host strain whichcontains a defined deletion along with an integrated suicide vector ischaracterized as a merodiploid, since it contains two different allelesof the same gene. Generally, the deletion constructed on the vector willrepresent a gene deletion and the integrated product on the chromosomewill have the structure characterized by the presence of a wild-typeallele flanking one end of the integrated vector, and the defineddeletion mutant allele at the other end of the vector. Otherconstructions are well known in the art.

Bacteria in which the suicide vector has been excised from thechromosome along with the antibiotic-resistance marker can be selectedon specialized media. Two such counter selection methods have beenemployed to identify these antibiotic-sensitive strains. One method,which is described in Example 1, relies on fusaric acid sensitivity oftetracycline resistant strains. Colonies which appear on fusaric acidplates are screened for the loss of tetracycline resistance and thepresence of the mutant allele. Another counter selection method takesadvantage of sucrose sensitivity using the sacRB system (Kaniga et al.,Gene 109:137-141, 1991) in which expression of levanosucrase in thepresence of 5% sucrose is toxic to cells retaining the sacB gene.

Following the introduction of any defined deletion mutant allele into astrain, phenotypes associated with the mutant gene are characterizedusing standardized tests well known in the art. These tests includedetermination of phenotypic reversion frequency, confirmation ofdeletion by Southern blot or PCR, agglutination by O-group specificantisera, production of complete LPS, presence of flagellar H antigen,motility, plasmid content and confirmation of auxotrophies.

Mutant strains may be shown by Southern blot to possess a loss ofgenetic material corresponding to the region deleted, as revealed by amobility shift of DNA relative to the wild-type and the defined deletionmutant allele constructed on a plasmid. PCR analysis of mutant strainssignificantly reduces the time required for confirming the presence ofdefined deletions since no DNA isolation is required and results can becompleted in less than one day. The PCR method also allows theidentification of erroneous recombination events or retention ofdelivery vector sequences, revealed as mobility shifts or the productionof multiple DNA fragments other than those expected upon gel analysis ofPCR products.

After construction, strains with defined deletion mutations are fullyevaluated for properties associated with the mutation and/or which areimportant for a strain to be immunogenic as well as attenuated. Forexample, production of full-length LPS similar to the parental wild-typestrain is evaluated using silver stained gels. The confirmation ofcorrect O-antigen is determined by antisera agglutination of mutantcells. Mutant strains are evaluated for positive agglutination usingdiluted poly H antiserum (Difco) and subjected to motility tests in softagar motility tubes relative to the parent strains and non-flagellatedcontrol strains, χ3420 and χ3422. Standard clinical API test strips areused following isolation of each mutant strain to obtain fermentationand biochemical data for comparison to parental strains. Growth ratesand plasmid content of the mutant strains are also compared to that ofparental strains. With S. typhi strains, the plasmid content is notevaluated because the large virulence plasmid present in S. typhimuriumis absent in S. typhi (Gulig et al., Infect. Immun. 56:3262-3271, 1987).

Construction of Defined Deletions in phoP, phoQ, and phoPQ Genes

The Salmonella phoPQ operon consists of phop and the adjacent downstreamphoQ genes. Defined deletions in the phoP and phoQ genes can beconstructed using an inverse PCR strategy since the entire nucleotidesequence of the operon and some flanking sequence is known. The DNAsequence reveals the presence and position of restriction sites whichcan be useful in constructing defined deletions in these genes. Thegenes can be isolated on a single 2,110 base pair PCR product and clonedinto a plasmid vector. The recombinant vector containing the phoPQ genecassette can be digested with restriction enzymes to delete most of thephoP gene, leaving the phoQ gene intact. The defined phoP deletion onthe phoPQ gene cassette can be inserted into a suicide vector, andintroduced into the chromosome of a wild-type phoPQ Salmonella toproduce an antibiotic-resistant merodiploid, which can be grown onappropriate media to select for the loss of the integrated plasmid alongwith the antibiotic-resistance marker. Antibiotic-sensitive strains canbe phenotypically characterized for the presence of an appropriatedefined deletion phoP mutant allele by screening for the loss of acidphosphatase activity using the agar overlay method of Kier et al. (J.Bacteriol. 130:399-410, 1997). A mutation in either phoP or phoQ issufficient to confer a PhoP⁻ phenotype.

Defined deletion mutants in phoQ or in both phoP and phoQ can begenerated using a similar strategy, using restriction enzymes to deletedefined segments of DNA from either phoQ or from both phoP and phoQ, andintroduced into the chromosome on a suicide vector to generatemerodiploids, which can be counter selected on appropriate media for theloss of the integrated plasmid and antibiotic-resistance marker, andphenotypically screened for the presence of the relevant defineddeletion mutant allele using PCR to verify the genotype.

Construction of Defined Deletions in the cya Gene

A recombinant vector which confers a maltose positive phenotype to an E.coli cya mutant strain when grown on MacConkey maltose media can be usedto construct a defined deletion in a Salmonella cya gene. Divergentprimers based on the known Salmonella cya gene sequence can be used inan inverse PCR reaction with the complementing recombinant vector as atemplate to generate a linear product consisting of the vector and DNAflanking either end of the deleted DNA specified by the PCR primerpositions. Alternatively restriction enzymes can be used to delete allor a portion of the complementing cya gene from the recombinant vector.

A defined deletion constructed using either method can be excised fromthe cloning vector using restriction enzymes and introduced into asuicide vector containing an antibiotic-resistance marker. The resultingrecombinant suicide vector containing the defined deletion cya allelecan be introduced into the chromosome of a wild-type cya Salmonellastrain to generate an antibiotic-resistant merodiploid. The merodiploidwould be grown on appropriate media to select for the loss of theintegrated plasmid along with the antibiotic-resistance marker.Antibiotic-sensitive strains would be phenotypically characterized forthe presence of an appropriate defined deletion Δcya-27 mutant allele.MGM-232 and χ8217 are two S. typhimurium UK-1 strains with definedΔcya-27 mutations that were constructed by these methods (see Table 1).

Construction of Defined Deletions in the crp Gene

Defined deletions in the Salmonella crp gene can be constructed using astrategy similar to that used for construction of a defined deletion incya. A recombinant vector can be selected which confers a maltosepositive phenotype to an E. coli crp mutant strain when grown onMacConkey maltose media. Divergent PCR primers can be used to delete theknown Salmonella crp gene and flanking sequences, and the resultingdefined deletion introduced into the chromosome of a wild-type crpSalmonella on a suicide vector to generate an antibiotic-resistantmerodiploid. The merodiploid could be grown on appropriate media toselect for the loss of the antibiotic resistance and the integratedplasmid, and antibiotic-sensitive strains could be phenotypicallycharacterized for the presence of an appropriate defined deletion crpmutant allele.

Construction of Δcya Δcrp Double Mutants

Strains which contain defined deletion mutations in both cya and crp canalso be constructed. For example, cya mutants can be constructed asdescribed above, and then a defined deletion mutant crp allele can beintroduced on a suicide vector to generate merodiploids selected on anappropriate antibiotic medium. Growth of merodiploids on an appropriatemedium such as fusaric acid or 5% sucrose as described above can be usedto counter select for the loss of the suicide vector along with theantibiotic-resistance gene from the chromosome, and an appropriatedefined deletion crp mutant can be phenotypically identified onMacConkey maltose medium containing 2 mM cAMP. This is because cAMPwhich is the product of adenylate cyclase encoded by the cya gene causesa cya mutant to be phenotypically Cya⁺ on MacConkey maltose agar.However, a crp mutation renders the cya mutant strain no longer capableof fermenting maltose and thus selection on maltose medium allows easydetection of strains with both of the defined Δcya and Δcrp mutations.MGN-431 and χ8214 are two strains with defined cya and crp deletionmutations constructed in this way (see Table 1).

Construction of Defined Deletions in pmi and cdt Genes

Mutations in other genes have also been shown to confer an attenuationon Salmonella, including cdt and pmi alleles. Defined deletions in thepmi gene can be constructed using an inverse PCR strategy, orrestriction enzymes. A recombinant vector which confers amannose-positive phenotype to an E. coli or Salmonella mutant pmi strainwhen grown on MacConkey mannose media can be used to construct a defineddeletion mutant pmi allele using either restriction enzymes or inversePCR. The defined deletion pmi allele can be inserted into a suicidevector and integrated into the chromosome of a pmi⁺ Salmonella togenerate an antibiotic-resistant merodiploid, which can be grown onappropriate media to select for the loss of the integrated plasmid alongwith the antibiotic-resistance gene. Antibiotic-sensitive strains wouldbe phenotypically characterized for the presence of an appropriatedefined deletion pmi mutant allele by screening for a reversiblerough-smooth phenotype, detecting a smooth phenotype due to thesynthesis of LPS O-antigen repeats when grown in the presence ofmannose, and a rough phenotype when grown in the absence of mannosedetected by the absence of agglutination in the presence of LPSO-antisera.

A defined deletion which confers a Cdt⁻ phenotype upon Salmonella can beconstructed using restriction enzymes to delete DNA associated with thisphenotype from a recombinant vector which complements a Salmonella cdtmutant. The mutant allele constructed using this strategy can beinserted into a suicide vector and introduced into the chromosome of acdt⁺ Salmonella to generate antibiotic-resistant merodiploids.Merodiploids can be grown on appropriate media to select fdr the loss ofthe integrated vector along with the antibiotic-resistance marker, andantibiotic-sensitive strains can be phenotypically characterized for thepresence of an appropriate defined deletion cdt mutant allele.

EXAMPLE 3

This example illustrates the construction of defined ΔphoPQ23 andΔasdA16 deletions in S. typhi ISP1820 and Ty2 to produce strainsMGN-1191 and MGN-1256, respectively.

The construction of the defined ΔphoPQ23 ΔasdA16 S. typhi strains inboth the ISP1820 and Ty2 backgrounds involved the use of two suicideplasmids, pMEG-213 containing the ΔphoPQ23 region and pMEG-006containing the ΔasdA16 region.

The ΔphoPQ23 deletion was obtained by digesting pMEG-068 with EcoRV andTthIIII1 removing the 1103 bp EcoRV-TthIII1 fragment encoding the Cterminal end of PhoP and the His region of PhoQ, responsible forphosphorylation of PhoP (FIG. 6). The linearized plasmid was thentreated with T4 DNA polymerase and religated to produce pMEG-210 (FIG.6). The BamHI-XbaI fragment of pMEG-210 containing the ΔphoPQ23 deletionwas then inserted in the pir-dependent suicide vector pMEG-149 toproduce pMEG-213 (FIG. 6). Since pMEG-213 is a mobilizable suicidevector encoding for the selectable marker for ampicillin resistance andthe counter-selectable marker, levanosucrase, resulting in sensitivityto sucrose, the plasmid can be conjugated into any strain desiredselecting for ampicillin resistance followed by counter-selection forthe replacement of the wild-type phoPQ genes with the mutant phoPQ23 inthe presence of sucrose. The host strain responsible for the delivery ofpMEG-213 was obtained by transforming pMEG-213 into the Pir⁺ Asddelivery host MGN-617 to produce MGN-758.

Introduction of the ΔphoPQ23 deletion into S. typhi ISP1820, χ3744, andS. typhi Ty2, χ3769, was accomplished by conjugating MGN-758 with thewild-type S. typhi parents and selecting for ampicillin-resistantisolates which grew without DAP (diaminopimelic acid). The isolatesobtained from this conjugation represent the first integration of theΔphoPQ23 deletion into the chromosome producing a duplication of thewild-type gene with the mutant ΔphoPQ23. These isolates, MGN-1037 andMGN-1017, were then plated on Luria agar with 5% sucrose to select forloss of the ampicillin-resistant suicide vector. The isolates obtainedby this selection were then screened for acid phosphatase activity usingthe agar overlay method of Kier et al. (J. Bacteriol. 130:399-410,1977). The white phosphatase-negative colonies were then confirmed forthe ΔphoPQ23 phosphatase minus phenotype and stocked as MGN-1038 for S.typhi ISP1820 and MGN-1018 for S. typhi Ty2.

The introduction of a defined asd deletion was then needed to provide avaccine strain for the delivery of heterologous antigens. The definedasd deletion present on pMEG-006 was obtained by performing inverse PCRon pMEG-003 to remove the majority of the coding region of the asd genebetween 219 and 1460 bp to produce pMEG-006 containing a new uniqueBglII site (FIG. 7). pMEG-006 has a pir-dependent replicon and thetetracycline-resistance gene from the transposon Tn10, but has nomobilization functions to allow conjugation.

Introduction of the defined ΔasdA16 deletion into any strain requiresplasmid DNA to be electroporated into the strain desired followed byselection for tetracycline resistance. The tetracycline-resistantisolates obtained can then be plated on fusaric acid containing media toselect for loss of the tetracycline-resistant elements of the suicidevector (Maloy et al., J. Bacteriol. 145:1110-1112, 1981) followed byscreening for the Asd⁻ DAP-requiring phenotype. Both MGN-1038 andMGN-1018 were electroporated with pMEG-006 and tetracycline-resistantisolates obtained. These isolates were then plated on fusaric acidplates containing 50 μg DAP/ml and the isolated colonies obtainedscreened for the loss of the tetracycline-resistance element of thesuicide vector and replacement of the wild-type asd gene with theΔasdA16 mutation. Isolates were then confirmed for tetracyclinesensitivity and requirement of DAP. An S. typhi ΔphoPQ23 ΔasdA16derivative of each strain was selected for further work, which aredesignated herein as MGN-1191 (ISP1820) and MGN-1256 (Ty2) (See Table1).

EXAMPLE 4

This example illustrates methods for testing Salmonella and otherEnterobacteriaceae strains for an RpoS⁺ phenotype.

Testing for catalase activity provides one method for determining therpoS allelic state of strains since RpoS positively regulates expressionof hydroperoxidase II catalase from the katE gene in S. typhimurium.(Lowen, supra). Cultures can be analyzed for catalase production as anindicator of the RpoS phenotype, by adding 100 μl of hydrogen peroxide(H₂O₂) to one milliliter of each strain grown to stationary phase(Mulvey et al., Gene 73:337-345, 1988). Vigorous bubbling of astationary phase culture after addition of H₂O₂ would suggest that thestrain contained a wild-type rpoS allele, whereas minimal bubbling wouldimply that the strain contained a defective rpoS allele.

The RpoS phenotype of attenuated Salmonella strains can also bedetermined by assessing the sensitivity of the strains to nutrientdeprivation and acid and oxidative stress. Salmonella ropS mutants areknown to have a reduced ability to survive these stresses as compared towild-type parents (Fang et al., Proc. Natl. Acad. Sci. 89:11978-11982,1992). Evaluation of prolonged stationary-phase survival can beperformed in M9 medium on a rotary shaker at 37° C. for 6 days.Susceptibility to pH 4.0 can be determined by pelleting stationary-phasebacteria and resuspending cells in L-broth adjusted to pH 4.0 withcitrate buffer. Sensitivity of strains to oxidative stress can bedetermined by the addition of hydrogen peroxide to stationary-phasebacteria in L-broth to a final concentration of 15 mM. In each of theseexperiments, bacteria are removed at timed intervals, eluted and platedonto L-agar for quantitation of viable cells.

The response of an S. typhimurium rpoS mutant strain to each of theabove mentioned stresses has been reported and compared to its virulentparent strain (Fang et al., supra). Specifically, the rpoS S.typhimurium mutant exhibited a 7-fold reduced ability to survive aprolonged. stationary phase relative to its wild-type parent in M-9media on a rotary shaker at 37° C. for 6 days. Likewise, the rpoS S.typhimurium mutant was 10-fold more susceptible to killing by pH 4.0 ascompared to its wild-type parent after stationary-phase cultures ofthese strains were resuspended in L-broth adjusted to pH 4.0 withcitrate buffer. In addition, 98% of an inoculum of the ropS S.typhimurium mutant did not survive a 60 minute exposure to 15 mM H₂O₂,whereas the wild-type parent was unaffected by this treatment.

Another approach that can be used to determine the RpoS phenotypeinvolves assessing the ability of the Salmonella to synthesize glycogen.It has been shown that rpoS null mutations result in a glycogen-negativephenotype (Lang et al., Mol Microbiol. 5:49-59, 1991). Specifically, theglgs gene is an ropS-dependent gene which is involved in glycogensynthesis. Furthermore, since a null glgS mutant accumulates moreglycogen than a rpoS mutant, rpoS may have further effects on glycogensynthesis in addition to glgs induction. Thus, it is possible todetermine the rpoS allelic state of strains by analyzing their abilityto accumulate glycogen.

Accumulation of glycogen is tested by growing cells as single coloniesor patches on Q-3 medium which contains 0.06M K₂HLPO₄, 0.03M KH₂PO₄,0.008M (NH₄)₂SO₄, 0.0017M sodium citrate, 8.1×10⁻⁴M MgSO₄, 1.9×10⁻⁴histidine HCl, 1.5×10⁻⁵M thiamine HCl, and 0.056M glucose. Whenanalyzing the glycogen biosynthesis abilities of auxotrophic strains,the appropriate nutrient supplements are added to Q-3 media. Forexample, methionine (20 μg/ml), threonine (80 μg/ml), leucine (20μg/ml), and DAP (100 μg/ml) must be added to Q-3 media to sustain thegrowth of Δasd mutant strains. Furthermore, since S. typhi Ty2 andISP1820 are cys and cys trp mutants, respectively, Q-3 media must besupplemented with each of these amino acids at a concentration of 20μg/ml. After about 20 hours of growth at 37° C., the cells are treatedwith a solution of iodine and potassium iodide. Strains which arewild-type and functional with respect to glycogen biosynthesis turnbrown after iodine treatment, while those strains which are defective inglycogen biosynthesis stain yellow.

In a variation of the above approaches, the RpoS phenotype can bedetermined by first moving the rpoS allele into either a wild-type S.typhimurium such as χ3339 if the allele to be tested is expected to havea ropS mutation or into the ropS mutant χ4973 if the allele is expectedto be rpoS⁺ using P22HTint-mediated transduction followed by subsequenttesting of the derived microbe for RpoS phenotype by any of the tests asdescribed above. Final proof of the allelic state of an ropS allele canbe achieved by DNA sequencing using PCR methods.

EXAMPLE 5

This example illustrates the superior ability of attenuated S.typhimurium rpoS⁺ strains having attenuating mutations in the cya, aroA,or in both the cya and crp genes in colonizing Peyer's patches of theGALT, compared to the colonizing ability of corresponding rpoS mutant S.typhimurium strains.

The attenuated rpoS⁺ S. typhimurium strains tested were MGN-431, aΔcya/Δcrp mutant; χ3679, an ΔaroA mutant; and MGN-232, a Δcya mutant(see Table 1 for derivations). Comparative S. typhimurium strainscontaining an inactive rpoS allele were constructed as described belowto obtain χ8214, χ8215 and χ8217.

Cultures were maintained as frozen cultures suspended in 1%Bacto-peptone containing 5% glycerol and fast-frozen in dry-ice ethanolfor storage in duplicate at −70° C. and also suspended in 1%Bacto-peptone containing 50% glycerol for storage at −20° C. for routineuse.

Complex medium for routine cultivation of S. typhimurium strains was Lbroth as described above. Difco agar was added to Lennox broth at 1.5%for base agar and 0.65% for soft agar. L agar was used for routineenumeration of bacteria. Fermentation was evaluated by supplementingMacConkey base agar (Difco, Detroit, Mich.) with 1% final concentrationof lactose.

In generating comparative rpoS mutant cultures, media were supplementedwith ampicillin (50 μg/ml) to select for ampicillin-resistant S.typhimurium strains containing the inactive ropS allele. Buffered salinewith gelatin (BSG) (Curtiss, 1965 supra) was used routinely as adiluent.

Bacteriophage P22HTint propagated on SF1005 was used to transduce theropS mutant allele into MGN-431, χ3679, and MGN-232 to generate χ8214,χ8215, and χ8217, respectively (see Davis et al., supra). An overnightculture of the donor strain was diluted 1:20 into prewarmed L broth,grown for 60 minutes with shaking at 37° C. and then infected withP22HTint at a multiplicity of 0.01. The infection mixture was shakenovernight for approximately 15 h, chloroform added and allowed to shakean additional 10 minutes at 37° C., and the suspension centrifuged(Sorvall RC5C, SS-34 rotor, 7,000 rpm, 10 min) to remove bacterialdebris. The supernatant fluid containing the phage (ca. 10¹⁰/ml) wasstored at 4° C. over chloroform. Ampicillin to a concentration of 50μg/ml was used to select for transductants containing an inactive rpoSallele.

The RpoS phenotype of the S. typhimurium strains was determined bytesting for catalase and glycogen synthesis activities as described inExample 4. Results are shown in Table 8.

TABLE 8 Catalase and Glycogen Activity Tests on S. typhimurium Strains.Glycogen Catalase Synthesis Strain Relevant Genotype Activity Activityχ3339 SL1344 PStSL100⁺ hisG rpsL, + + colicin⁺ χ4973 SL1344 pStSR100⁺hisG rpsL, − − rpoS::RR10, colicin⁺ χ3761 UK-1 wild-type prototroph + +χ4937 UK-1 rpoS::RR10 − − MGN-232 UK-1 Δcya-27 + + χ8217 UK-1 rpoS::RR10Δcya-27 − − MCN-431 UK-1 Δcya-27 Δcrp-27 + − χ8214 UK-1 rpoS::RR10Δcya-27 − − Δcrp-27

Those Salmonella known to have a wild-type rpoS gene showed catalaseactivity, whereas, those strains having a mutation in the rpoS geneshowed no catalase activity. Results with glycogen activity testingagreed with catalase testing with the exception that MGN-431, which hasan rpoS gene and was catalase positive, nevertheless, gave negativeresults in the glycogen test. This is undoubtedly due to the fact thatglycogen synthesis is also dependant on crp gene function.

Female BALB/c mice (6 to 10 weeks old) (Charles River Laboratories,Wilmington, Mass.) were used for infectivity and/or immunizationexperiments. Animals were held for one week in a quarantined room priorto being used in experiments. Experimental mice were placed in Nalgenefilter-bonnet-covered cages with wire floors. Food and water werewithheld for 4-6 hours prior to peroral infection.

The animal infectivity of S. typhimurium strains was determinedfollowing peroral (p.o.) inoculation. Bacteria for inoculation in micewere grown overnight as standing cultures at 37° C. in L broth. Thesecultures were diluted 1:200 into prewarmed broth and aerated at 37° C.for approximately 4 h to an OD₆₀₀ of 0.8. The cells were concentrated50-fold by centrifugation in a GSA rotor at 7,000 rpm for 10 min at 4°C. in a Sorvall RC5C centrifuge followed by suspension in BSG. Suitabledilutions were plated on L agar for titer determination. For all p.o.inoculations with S. typhimurium, mice were deprived of food and waterfor 4-6 h prior to inoculation. They were then fed 20 μl of S.typhimurium suspended in BSG using a Pipetman P20. Food and water werereturned 30 minutes after oral inoculation.

In order to assess the colonization of the GALT and, in particular,Peyer's patches, by rpoS⁺ attenuated S. typhimurium strains, threegroups of three mice each were inoculated perorally with equal numbers(approximately 10⁹ CFU) of an rpoS⁺ attenuated S. typhimurium strain andits corresponding rpoS::RR10 mutant derivative, which were grownaccording to the conditions described above. Quantitation of viable S.typhimurium in Peyer's patches was performed as follows. The mice wereeuthanized at 3, 5, and 7 days after p.o. infection and their Peyer'spatches collected. The Peyer's patches from each mouse were asepticallyremoved and placed in polypropylene tubes with BSG, homogenized with aBrinkmann tissue homogenizer (Brinkmann Instruments) and placed on ice.Appropriate dilutions of the homogenate were plated on MacConkey agarsupplemented with lactose at 1% with and without ampicillin.Differentiation of the strains was facilitated by the presence of anampicillin-resistance marker within the inactive rpoS::RR10 allele.Plates were incubated for 12-15 hours at 37° C. Titers in the respectivePeyer's patches were determined for each time period and the geometricmeans calculated for 3 mice per group at each time of sampling.

Table 9 below shows the distributions of rpoS⁺ and rpoS::RR10 mutant S.typhimurium strains containing Δcya/Δcrp, ΔaroA, or Δcya mutations inmurine Peyer's patches after peroral infection.

TABLE 9 Geometric mean ratios of attenuated rpoS⁺ S. typhimuriumΔcya/Δcrp, ΔaroA, or Δcya strains to their corresponding isogenicrpoS::RR10 mutant Δcya/Δcrp, ΔaroA or Δcya derivatives in murine Peyer'spatches after peroral coinfection^(a) Time after in- rpoS⁺ Δcya/ΔcrprpoS⁺ ΔaroA rpoS⁺ Δcya fection rpoS::RR10 Δcya/Δcrp rpoS::RR10 ΔaroArpoS::RR10 Δcya 3 days 2.1 ± 0.7 1.2 ± 0.2 6.5 ± 2.6 5 days 1,468 ±1,271 4.3 ± 3.0 1.7 ± 0.8 7 days 308 ± 196 4,135 ± 4,132 7.1 ± 4.2^(a)Approximately equal numbers of MGN-431 (rpoS⁺ Δcya/Δcrp) and χ8214(rpoS::RR10 Δcya/Δcrp) (5.2 × 10⁸ and 5.4 × 10⁸, respectively); χ3679(rpoS⁺ ΔaroA) and χ8215 (rpoS::RR10 ΔaroA) (6.0 × 10⁸ and 6.0 × 10⁸ ,respectively); or MGN-232s (rpoS⁺ Δcya) and χ8217 (rpoS::RR10 Δcya) (6.8× 10⁸ and # 6.4 × 10⁸, respectively), were administered p.o. to8-week-old BALB/c mice. Geometric mean ratios ± S.E.M. are given (n =3).

The rpoS⁺ S. typhimurium strain containing Δcya/Δcrp mutations, MGN-431,exhibited a significantly greater ability to colonize Peyer's patches at5 days after oral infection compared to its rpoS::RR10 derivativestrain, χ8214. At 3 and 5 days after oral infection, the rpoS⁺ AaroA S.typhimurium strain χ3679 and its rpoS::RR10 derivative, χ8215, did notexhibit any significant differences in ability to colonize the Peyer'spatches. However, by 7 days postinfection, the rpoS::RR10 ΔaroA mutantdisplayed a significantly lower ability to colonize Peyer's patches ascompared to its ΔaroA parent strain, χ3679.

Coynault et al. have also reported that rpoS ΔaroA derivatives aredefective in colonizing murine Peyer's patches compared to rpoS⁺ parentstrains, however, the decrease in colonization was observed at theearlier times of 2 and 5 days after oral infection compared to thedecrease in colonization at 7 days reported here. (Coynault et al., Mol.Microbiol. 22:149-160, 1996).

Similar studies were done with Δcya mutants. As shown in Table 9, whenadministered orally to mice in approximately a 1:1 ratio, the rpoS::RR10Δcya mutant strain χ8217 exhibited a reduced ability to colonize Peyer'spatches at 3 and 7 days (ca. 6 and 7 fold, respectively) as compared toits parent strain, MGN-232.

EXAMPLE 6

This example illustrates the superior balance of high immunogenicity andlow virulence of the rpoS⁺ S. typhimurium strains of Example 5 havingeither aroA or cya mutations, compared to that of the correspondingisogenic rpoS⁻ mutant S. typhimurium strains.

Protective immunity elicited by attenuated S. typhimurium strains havingan rpoS⁺ genotype compared to the corresponding rpoS mutant strains wasdetermined in BALB/c mice following peroral inoculation as follows. Fivemice per group were p.o. inoculated with 10⁶, 10⁷, 10⁸ and 10⁹ CFU ofthe attenuated S. typhimurium rpoS⁺ strain or its isogenic ropS mutantderivative, respectively. Four weeks after immunization, mice werechallenged p.o. with 10⁹ CFU of the wild-type SR-11 or UK-1 virulentparent strain. The degree of protection is determined by the number ofmice alive 30 days after challenge and the data are shown in Tables 10and 11 below.

TABLE 10 Protection in mice against challenge with the virulentwild-type SR-11 strain (χ3181) after immunization with (A) χ3679 (rpoS⁺ΔaroA) or (B) its isogenic rpoS mutant derivative, χ8215 ImmunizationChallenge Dose Live/Total Dose^(a) of SR-11^(a) (%) A. χ3679 1.6 × 10⁶ 1× 10⁹ 2/5 (40%) 1.6 × 10⁷ 1 × 10⁹ 2/5 (40%) 1.6 × 10⁸ 1 × 10⁹ 4/5 (80%)1.6 × 10⁹ 1 × 10⁹ 4/5 (80%) B. χ8215 1.4 × 10⁶ 1 × 10⁹ 0/5 (0%)  1.4 ×10⁷ 1 × 10⁹ 1/5 (20%) 1.4 × 10⁸ 1 × 10⁹ 3/5 (60%) 1.4 × 10⁹ 1 × 10⁹ 3/5(60%) ^(a)Data represented as colony forming units per ml.

TABLE 11 Protection in mice against challenge with the virulentwild-type UK-1 strain (χ3761) after immunization with (A) MGN-232 (rpoS⁺Δcya) or (B) its isogenic rpoS mutant derivative, χ8217 ImmunizationChallenge Dose Live/Total Dose^(a) UK-1^(a) (%) A. MGN-232 2.0 × 10⁶ 8 ×10⁸ 4/5 (80%) 2.0 × 10⁷ 8 × 10⁸  5/5 (100%) 2.0 × 10⁸ 8 × 10⁸  5/5(100%) 2.0 × 10⁹ 8 × 10⁸  5/5 (100%) B. χ8217 1.2 × 10⁶ 8 × 10⁸ 2/5(40%) 1.2 × 10⁷ 8 × 10⁸ 1/5 (20%) 1.2 × 10⁸ 8 × 10⁸ 2/5 (40%) 1.2 × 10⁹8 × 10⁸ 2/5 (40%) ^(a)Data represented as colony forming units per ml.

The data presented in Table 10 indicate that, regardless of the doseused for vaccination, mice orally immunized with the rpoS⁺ ΔaroA mutant,χ3679 were better protected against oral wild-type challenge than weremice immunized with the isogenic rpoS mutant strain, χ8215. Similarly,Table 11 shows that immunization with rpoS⁺ microbes attenuated with aΔcya mutation provided better protection against the wild-type challengethan immunization with the isogenic rpoS mutant derivative.

Thus, this study shows that a S. typhimurium strain having a functionalrpoS gene provides protective immunity that is significantly better thanthat of the isogenic rpoS mutant strain when challenged orally with thewild-type virulent Salmonella strain. Thus, the presence of a functionalrpoS allele in S. typhimurium increases the immunogenicity of the strainto facilitate the stimulation of a high level of protective immunity.

EXAMPLE 7

This example illustrates the superior immunogenicity of an attenuatedRpoS⁺ strain of S. typhimurium following intranasal administrationcompared to the immunogenicity of the corresponding RpoS⁻ strainadministered by the same route.

Bacteria for intranasal immunization in mice were grown overnight asstanding cultures at 37° C. in L broth. The following morning, thesecultures were diluted 1:200 into L broth and aerated at 37° C. untilreaching an OD₆₀₀ of 0.8. The cells were concentrated by centrifugationin a Sorvall GSA rotor at 7,000 rpm for 10 min at 4° C. followed bysuspension in BSG. Suitable dilutions were plated on L agar for titerdeterminations.

For each attenuated bacterial vaccine strain, intranasal immunizationswere performed with eight-week-old female BALB/c mice such that eachmouse received either 10⁹ or 10⁸ cfu in a total volume of 0.01 ml (10μl) of BSG using a micropipette to administer droplets into one or bothnostrils. Immunization was accomplished by inoculating each nostril with0.005 ml (5 μl) of suspension or one nostril with 0.01 ml (10 μl) ofsuspension, or in the case of the controls with BSG lacking anybacteria. Food and water were returned within 30 min followingintranasal immunization.

Intranasally immunized mice and non-immunized controls were orallychallenged with either 10⁸ or 10⁹ cfu of the wild-type virulent S.typhimurium strain, χ3339, 30 days after the date of intranasalimmunization. The χ3339 challenge strain was grown overnight as astanding culture at 37° C. in L broth. The following morning the culturewas diluted 1:200 into L broth and aerated at 37° C. until reaching anOD₆₀₀ of 0.8. The cells were concentrated by centrifugation in a SorvallGSA rotor at 7,000 rpm for 10 min at room temperature followed bysuspension in BSG. The mice to be perorally challenged were deprived offood and water for approximately 4 h prior to the oral challenge. Micewere observed over a period of 30 days for morbidity and mortality. Thedata from this experiment are reported in Table 12.

TABLE 12 Effectiveness of intranasal iminunization with S. typhimuriumSL 1344 Δcya Δcrp RpoS⁺ vs. Δcya Δcrp RpoS⁻ mutants in protecting femaleBALB/c mice against peroral challenge with wild-type strain χ3339^(a)Immunizing Challenge Survivors/ Strain Genotype dose (CFU) dose (CFU)total χ8296 Δcrp-28 1.2 × 10⁹ 1.1 × 10⁹ 1/4 Δcrp-27 1.1 × 10⁸ 3/4ΔasdA16 1.2 × 10⁸ 1.1 × 10⁹ 0/4 RpoS⁺ 1.1 × 10⁸ 1/4  5/16 χ8309 Δcrp-281.5 × 10⁹ 1.1 × 10⁹ 0/4 Δcrp-27 1.1 × 10⁸ 1/4 ΔasdA16 1.5 × 10⁸ 1.1 ×10⁹ 1/4 rpoS RpoS− 1.1 × 10⁸ 0/4  2/16 None BSG 1.1 × 10⁸ 0/4 ^(a).Strains were grown in Luria broth with DAP. Preparation of bacterialinocula and animal infection were done as described in text. Oralchallenge with S. typhimurium χ3339 was given thirty days after intranasal immunization. Mortality was monitored for thirty days afterchallenge.

As shown in the table, intranasal administration of both the RpoS⁺microbe (χ8296) and the RpoS⁻ microbe (χ8308) provided some protectionagainst challenge by the wild-type strain (χ3339). The RpoS⁺ strain wasmore effective, however, in that this strain provided greater protectionagainst challenge with the wild-type strain (5 out of 16 survivors) thandid the corresponding RpoS⁻ strain (2 out of 16 survivors).

The experiment in this example utilized Δcya Δcrp Δasd strains of S.typhimurium that were either RpoS⁺ (χ8296) or RpoS⁻ (χ8309). Thesemicrobes did not contain an Asd⁺ plasmid vector which would functionallyreplace the chromosomal Δasd mutation so that they would be expected todie due to inability to synthesize diaminopimelic acid, within the first24 hours after intransal immunization. This would, in turn, be expectedto diminish the immunologic response that would have been elicited bythe microbes had they been endowed with an Asd-containing plasmid thatwould normally be incorporated into a vaccine microbe. Nevertheless, asnoted above, 5 of 16 mice immunized with the RpoS⁺ strain, χ8296,survived challenge whereas only 2 of 16 mice intranasally immunized withthe RpoS⁻ strain, χ8309, survived oral challenge with the wild type S.typhimurium strain, χ3339. Thus, even during the first 24 hours afteradministration, the RpoS⁺ strain showed a superior ability to elicit aprotective immune response.

The experiment described above was repeated using derivatives of χ8296(RpoS⁺) and χ8309 (RpoS⁻) that had been modified by introducing the Asd⁺plasmid vector pYA3342 by electroporation. These strains would,therefore, not die due to DAPless death. The strains were grown the sameway and the mice were immunized intranasally as described above althoughthe doses used were reduced to ˜3×10⁷ CFU and ˜3×10⁸ CFU. As revealed bythe data presented in Table 13, all 18 of 18 mice immunized intranasallywith χ8296 (pYA3342), the RpoS⁺ strain, survived oral challenge withwild-type χ3339 whereas only 17 of 20 mice immunized intranasally withthe χ8309(pYA3342), the RpoS⁻ strain, survived oral challenge withχ3339.

TABLE 13 Effectiveness of intranasal immunization with S typhimuriumSL1344 Δcya Δcrp Δasd RpoS⁺ vs Δcya Δcrp Δasd RpoS⁻ strains containingpYA3342 in protecting mice again p.o. challenge with wild-type χ3339*.Immunizing Challenge dose dose Survivors/ strain Genotype (CFU) (CFU)total χ8296 Δcya 3.7 × 10⁸ 9.6 × 10⁶ 4/4 (pYA3342) Δcrp 9.6 × 10⁷ 4/4Δasd 3.7 × 10⁷ 9.6 × 10⁸ 5/5 (RpoS⁺) 9.6 × 10⁷ 5/5 (18/18) χ8309 Δcya3.5 × 10⁸ 9.6 × 10⁸ 5/5 (pYA3342) Δcrp 9.6 × 10⁷ 5/5 Δasd 3.5 × 10⁷ 9.6× 10⁸ 3/5 rpoS 9.6 × 10⁷ 4/5 (RpoS⁻) (17/20) *Four weeks after mice wereimmunized I.N. with a single dose of the strains, they were challengedP.O. with wild-type SL1344 strain χ3339. Morbidity and mortalityobservations were recorded daily for an additional 30 dayspostchallenge. Both inoculating and challenge doses were measured inCFU.

As has been previously reported in the literature, recombinantattenuated Salmonella vaccine strains can be administered by variousroutes to stimulate mucosal and systemic immunity. For example,Srinivasan et al. (Vaccines 95, R. N. Chanock et al., Eds., Cold SpringHarbor Laboratory Press, Plainview, N.Y., p 273-280, 1995) and Hopkinset al. (Infect Immun. 63:3279-3286, 1995) reported that mice can beimmunized not only perorally and intragastrically, but alsointranasally, intravaginally and rectally. Nardelli-Haefliger et al.(Infect Immun 64:5219-5224, 1996) demonstrated that human volunteerscould be immunized rectally with a recombinant attenuated Salmonellatyphi vaccine strain. More recently, Galan et al. (Vaccine 15:700-708,1998) demonstrated that recombinant attenuated S. typhi Ty2 strains ofan RpoS⁻ phenotype are able to elicit immune responses when intranasallyadministered to mice. It is well known that M cells overlie epitheliallymphoid tissues not only in the small intestine (the so-called Peyer'spatches which are part of the GALT) but also in the rectum, in the CALT,in the BALT and possibly in other inductive sites leading to mucosalimmune responses (Mucosal Immunology, 2nd Edition, Ogra et al., Eds.Academic Press, San Diego, 1999). The examples above demonstrated thatRpoS⁺ Salmonella invade and collonize epithelial dome M cells in Peyer'spatches of the GALT and elicit an immune response followingadministration by the oral route. This example shows that an immuneresponse is also elicited upon administration by the intranasal route.On the basis of these results, it is logical to infer that RpoS⁺Salmonella are better able to attach to and invade M cells overlyinglymphoid tissues in the upper respiratory tract as well as to the Mcells of the GALT than are Salmonella strains that are defective withrespect to expression of the rpoS⁺ gene (i.e., are RpoS⁻ in phenotype).It, therefore, follows that immunization of humans with recombinantattenuated Salmonella vaccines displaying an RpoS⁺ phenotype would bemore efficacious than those displaying a RpoS⁻ phenotype. Therefore,RpoS⁺ attenuated Salmonella would be superior to RpoS⁻ attenuatedSalmonella for intranasal, oral, intragastric and rectal immunization.Since administration of attenuated Salmonella expressing foreignantigens to colonize mucosal lymphoid tissues is of paramount importancein eliciting mucosal immunity, it follows that such can be accomplishedby use of RpoS⁺ attenuated Salmonella of any of various serotypes notonly including S. typhi but S. paratyphi A, S. paratyphi B, and S.paratyphi C, which are also restricted to humans, but also attenuatedderivatives of such other serotypes of S. enterica such as Typhimurium,Enteritidis, Dublin, and Choleraesuis.

EXAMPLE 8

This example illustrates the superior ability of RpoS⁺ recombinantattenuated Salmonella vaccines to induce mucosal IgA and serum IgGantibodies to an expressed foreign antigen compared to that of thecorresponding RpoS⁻ Salmonella.

For these studies, the S. typhimurium strains used were attenuated withΔcya, Δcrp and Aasd mutations and were of either an RpoS⁺ phenotype(χ8296; Table 1) or an RpoS⁻ phenotype (χ8309; Table 1). Both of thesestrains were genetically engineered to produce the hepatitis B viruscore (HBVc) particles with pre-S1, S2 fusions according to methodsreported in the literature (Schodel et al., Infect. Immun. 62:1669-1676,1994); from pYA3167 (Nardelli-Haefliger et al., supra, 1996). Theplasmid specifying the HBVc preS1, S2 fusion was electroporated intoχ8296 and χ8309 and the resulting strains were evaluated for productionof the HBVc particles with the preS1, S2 epitopes. FIG. 8A depictsCoomassie blue stained SDS gels whereas FIG. 8B depicts the results ofanalysis of gels by Western blot using a monoclonal antibody 2A42 fromHybridoma-5520 directed at the preS2 epitope. As shown in the figures,both constructs produced the fusion protein which is readily detectableon the Coomassie blue gels as well as following Western blot analysis.

To evaluate the relative immunogenicity of the two strains, groups offemale BALB/c mice (eight-weeks old) were perorally immunized with 10⁹cfu of the pYA3167-transformed vaccine strain derivatives of χ8296 andχ8309. According to the immunization schedule described by Schodel etal. (1994, supra), mice were immunized orally with two doses of vaccinegiven two days apart. Strains were grown in L broth as standingovernight cultures at 37° C. In the morning, 1:200 dilutions into Lbroth were grown with moderate aeration until achieving an OD₆₀₀ of 0.8.Bacteria were sedimented by centrifugation and suspended in BSG todesired densities so that the vaccine dose could be administered in avolume of 0.02 ml (20 μl). Food and water were withdrawn from the miceapproximately 5 h prior to peroral immunization and were returned 30 minafter immunization. Serum samples and vaginal washings were collected 4and 6 weeks after initial immunization (for methodology, see Zhang etal., Biol. Reprod. 56:33-41, 1997). Serum IgG antibody and IgA antibodyin vaginal washings were detected by ELISA measuring antibody to afull-length pre-S protein (histidine fusion).

The protocol for ELISA was as follows. Ninety-six-well Immulon-1 plates(Dynatech, Chantilly, Va.) were coated with 10 μg of recombinant HBVpre-S protein (awd)/ml in 0.2 M bicarbonate/carbonate buffer (pH 9.6) at4° C. overnight. Nonspecific binding sites were blocked with 1% BSA inphosphate buffered saline (PBS)+0.1% Tween20 (pH 7.4) (blocking buffer)at room temperature for 1 h. Serum samples and vaginal washings werediluted 1:100 and 1:10, respectively, in blocking buffer. One hundredmicroliters of the diluted samples were added in duplicate to the platesand incubated at 37° C. for 2 h. The plates were then washed withPBS+0.1% Tween20 three times. One hundred microliters of biotin-labelledgoat anti-mouse IgA or IgG were added, respectively, and incubated at 4°C. overnight. Alkaline phosphatase-labelled ExtrAvidin (Sigma) was addedto the plates and incubated at room temperature for 1 h. Substratesolution (0.1 ml) containing p-nitro-phenylphosphate (1 mg/ml) in 0.1 Mdiethanolamine buffer (pH 9.8) was added and the optical density of theresulting substrate reaction read at 405 nm with an automated ELISAreader (BioTech, Burlington, Vt.). All the reagents were purchased fromSigma (St. Louis, Mo.).

The results of the antibody titer determinations are in FIG. 9. As shownin the figure, the RpoS⁺ recombinant attenuated vaccine strain, χ8296,induced significantly higher antibody titers against the recombinantHBVc preS1, S2 antigen than did the corresponding RpoS⁻ microbe, χ8309,both in serum and in vaginal secretions at 4 and 6 weeks followingperoral immunization. It is, therefore, evident that recombinantattenuated Salmonella vaccine strains of an RpoS⁺ phenotype are not onlysuperior in inducing protective immunity against Salmonella as was shownin Example 7 above, but they are also superior in inducing immuneresponses against expressed foreign antigens.

A more extensive repetition of the experiment yielding the data in FIG.9, corroborated these results and revealed that the isogenic RpoS⁻vaccine strain, χ8309(pYA3167), not only induced lower IgG and IgAantibody titers to the HBV pre-S peptide than the RpoS⁺ vaccine strain,χ8296(pYA3167), (FIGS. 10A and 11A), but was also inferior in inducingIgG and IgA antibodies to the Salmonella LPS antigen (FIGS. 10B and 11B)

EXAMPLE 9

This example illustrates the method for screening for vaccine strainscontaining an RpoS⁺ phenotype.

The evaluation of strains for RpoS⁺ phenotype allows the identificationand selection of RpoS⁺ strains. Such strains would be expected to showhigh immunogenicity. Strains for testing in a screening system for RpoS⁺phenotype can be from any source. For example, strains obtained fromdepositories can be tested as illustrated below.

Testing for catalase activity and glycogen biosynthesis was performed asdescribed in Example 4 above.

The results of testing for catalase or glycogen synthesis activity intypical S. typhi strains are shown below in Table 14. Strains χ8205 andχ8208 did not show catalase activity which is consistent with earlierreports that these microbes are rpoS mutants (Robbe-Saule et al., FEMSMicrobiol. Lett. 126:171-176, 1995; Coynault et al., Mol Microbiol.22:149-160, 1996). The results of the catalase test suggest that strainsχ8204 and χ8207 may also have rpoS mutations, however, the glycogen testwas positive for these two strains suggesting that the two strains havean intact rpoS gene. A final decision as to the rpoS allelic state inthese two strains would, therefore, require use of other tests asdescribed in Example 4. The remaining strains for which results wereobtained in both the catalase and the glycogen test showed correspondingresults in both tests.

TABLE 14 Catalase and Glycogen Synthesis Tests on S. typhi strainsGlycogen Relevant Catalase Synthesis Source/ Strain GenotypeActivity^(a) Activity Reference χ3743 ISP1804 Type 46 ± + See Table 1χ3744 ISP1820 Type 46 + + See Table 1 χ3745 ISP2822 Type E1 + + SeeTable 1 χ3746 ISP2825 Type E1 + + See Table 1 χ3769 Ty2 Type E1 − − SeeTable 1 χ8203 cys, trp + + ATCC 9992V χ8204 cys, trp − + ATCC 33458χ8205 Ty21a galE, − No ATCC 33459 rpoS, cys, trp Growth χ8206 cys, trp,aroA + + ATCC 39926 serC, purA χ8207 cys, trp − + ATCC 10749 χ8208 Ty2cys, rpos − − ATCC 19430 χ8209 cys, trp + + ATCC 9993 MGN-1256 Ty2 rposcys − − See Table 1 ΔphoPQ23 ΔasdA16 MGN-1191 ISP1820 cys + + See Table1 trp ΔphoPQ23 ΔasdA16 ^(a)Vigorous bubbling upon addition of H₂O₂ isindicated by +, an intermediate level of bubbling is indicated by ±, andlittle or no bubbling is indicated by −.

EXAMPLE 10

This example illustrates a method that can be used to introduce awild-type ropS allele into RpoS⁻ S. typhi strains such as χ3769,MGN-1018 or χ8280 using an allelic replacement strategy.

The wild-type rpoS gene can be introduced into the chromosome of χ3769,MGN-1018 or χ8280 by allelic exchange using the suicide properties ofthe R6K-based plasmid pMEG-149 or its derivative pMEG-375. PlasmidspMEG-149 and pMEG-375 are mobilizable suicide vectors which carry aλpir-dependent R6K replicon and thus require a host with the pir genepresent in trans to allow replication. In addition, pMEG-149 encodes theselectable marker for Ap^(r) and the counterselectable marker,levanosucrase whereas pMEG-375 also contains the cat gene specifyingresistance to chloramphenicol (Cm^(r)). Since pMEG-149 and pMEG-375cannot replicate in strains lacking the pir gene, selection of Ap^(r)and Ap^(r) Cm^(r) transconjugants, respectively, demands the integrationof the plasmid into the chromosome, an event which usually takes placethrough homology in the inserted fragment.

Plasmid pSK::rpoS contains the entire 1.7 kb S. typhimurium 14028 rpoSgene cloned into the EcoRV site of pBluescript/SK. The EcoRI-HindIIIfragment containing the wild-type ropS allele from pSK::rpoS was treatedwith T4 DNA polymerase and cloned into the SmaI site of the suicidevector pMEG-149. The resulting recombinant vector carrying the wild-typeropS allele designated as pYA3433 (FIG. 12), would be introduced intothe λPir⁺ Asd⁻ delivery host strain, MGN-617. This strain allows theconjugal transfer of any plasmid containing an IncP mob region to anyAsd⁺ recipient, followed by elimination of the donor on any medialacking diaminopimelic acid (DAP).

Since duel selection for two drug-resistance genes often enhancesselection of merodiploid strains that have integrated a suicide vectorinto the chromosome by eliminating background growth that sometimesoccurs when using Ap^(r) alone due to the ability of β-lactamase torapidly destroy the ampicillin in the selective medium, we also made asuicide vector with the rpoS⁺ gene using pMEG-375 which specifieschloramphenicol resistance in addition to ampicillin resistance. The1.409 kb S. typhimurium UK-1 rpoS⁺ gene was recovered from pMEG-328 bydigestion with PmeI and SmaI and cloned into pMEG-375 digested with thesame two enzymes. The resulting suicide vector plasmid, pYA3467, isdepicted in FIG. 13.

Plasmid pYA3467 carrying the wild-type rpoS allele was introduced viaMGN-617 into the ΔphoPQ Δasd S. typhi Ty2 strain, MGN-1018, byelectroporation. Transformants were selected by spreading on L-agarplates supplemented with DAP (100 μg/ml), ampicillin (50 μg/ml) andchlormphenicol (40 μg/ml) followed by incubation overnight at 37° C.Ampicillin- and chloramphenicol-resistant isolates obtained from thistransformation procedure represent the integration of the entire plasmidincluding the wild-type rpoS allele into the chromosome by a singlecrossover event. Such isolates contain two copies of the rpoS gene, ie.a wild-type and a mutated rpoS allele. The isolates were then screenedon Luria agar supplemented with DAP (100 μg/ml) and containing 5%sucrose to select for loss of the suicide vector sequences by a secondcrossover event. Sucrose-resistant isolates were screened forsensitivity to ampicillin and chloramphenicol and for the presence of afunctional rpoS allele (using the catalase or glycogen synthesis test).

After identifying bonifide rpoS⁺ derivatives, complete characterizationis done to verify the presence of LPS, Vi antigen, all attenuatingmutations and the presence of all other traits that are characteristicof an RpoS⁺ derivative of the MGN-1018 parent. One such derivative wasselected as χ8434 (Table 1). Since the wild-type S. typhi, Ty2 strainmay possess excellent attributes as a recombinant attenuated Salmonellavaccine vector if provided with an RpoS⁺ phenotype, the wild-type Ty2strain χ3769 was also endowed with the rpoS⁺ gene from pYA3467 using themethod described above to generate the RpoS⁺ derivative χ8438 (ATCC202182)(Table 1). This strain can now be attenuated by introducingvarious defined deletion mutations as described in Examples 2 and 3 andthen endowed with the ability to express various antigens as describedin Example 11 below.

In order to further validate the method, pYA3467 was transferred by thedonor MGN-617 to the candidate recombinant attenuated vaccine strainχ8280 [Ty2 ΔphoPQ23 rpoS ΔasdA16 (pYA3167)]. This strain synthesizes thehepatitis B virus core with pre S1, S2 epitopes due to the presence ofpYA3167. Using the procedures described above, an RpoS⁺ derivative wasisolated and fully characterized. This was designated χ8435 (Table 1).

The abilities of these three RpoS⁺ strains, constructed by introducing arecombinant wild-type ropS⁺ gene to replace the defective rpoS mutantgene present in S. typhi Ty2 and its descendents, to synthesize glycogenand to give a positive catalase test are shown in Table 14.

TABLE 15 Test for RpoS Phenotype in Recombinant Salmonella Strains andTheir Parents. Glycogen Catalase Salmonella typhi Ty2Accumulation/Boisynthesis Activity χ3769 S. typhi Ty2 − − wild-typeχ8438 S. typhi Ty2 + + rpoS⁺ MGN-1018; S. typhi Ty2 − − ΔphoPQ23 χ8434S. typhi Ty2 + + ΔphoPQ23 rpoS⁺ χ8280 (pYA3167) − − S. typhi Ty2ΔphoPQ23 ΔasdA16 χ8435 (pYA3167) + + S. typhi Ty2 ΔphoPQ23 ΔasdA16 rpoS⁺

This method can also be used to introduce a recombinant wild-type rpoSgene into various Ty2 derived vaccine strains such as ATCC 55117 (χ3927;Δcya-12 Δcrp-11) or ATCC 55118 (χ4073; Δcya-12 Δ[crp-cdt]-10) to improvethe balance between attenuation and immunogenicity.

EXAMPLE 11

This example illustrates the construction of recombinant attenuatedrpoS⁺ S. typhi strains expressing foreign antigens for use as oralvaccines to immunize against various infectious diseases.

The rpoS⁺ vaccine strains are prepared based upon S. typhi strainscontaining a functional rpoS gene such as ISP1820 using defineddeletions as described above in examples 2 and 3 or based uponattenuated rpoS mutant strains such as Ty2 which have a recombinant rpoSgene as described in example 10 above. In the construction of vaccinesexpressing foreign antigens, the preferred approach is to use abalanced, lethal host-vector system which confers stable maintenance andhigh-level expression of cloned genes on recombinant plasmids. For this,a chromosomal mutation of the asd gene encoding aspartate β-semialdehydedehydrogenase is introduced into the RpoS⁺ strain to impose an obligaterequirement for diaminopimelic acid (DAP) which is an essentialconstituent of the rigid layer of the bacterial cell wall and which isnot synthesized in humans. The chromosomal Δasd mutation is thencomplemented by a plasmid cloning vector possessing the wild-type asd⁺gene as well as a recombinant gene encoding the desired foreign antigen.Loss of the plasmid results in DAP-less death and cell lysis. Suchbalanced-lethal host-vector combinations are stable for several weeks inthe immunized animal host and elicit immune responses against the clonedgene product as well as against Salmonella.

The construction of a defined deletion in the chromosomal asd gene isdescribed in example 3 above. The ISP1820 derivative, MGN-1191 and theTy2 derivative, MGN-1256, which have ΔphoPQ23 and ΔasdA16 mutations werethus produced. The asd-complementing plasmid containing a recombinantgene encoding the desired foreign antigen can be constructed asdescribed in U.S. Pat. No. 5,672,345. For example, one such plasmidexpressing the Hepatitis B virus antigenic nucleocapsid pre-S1 pre-S2(HBcAg-pre-S) particles, designated as pYA3167, has been constructed asreported in the literature (Schodel, et al., 1996, in Novel strategiesin design and production of vaccines. S. Cohen and A. Shafferman, eds.,Plenum Press, New York). Accordingly, S. typhi MGN-1191 and MGN-1256have been transformed with plasmid pYA3167 via electroporation.Immunoblot analysis with HBV pre-S2-specific monoclonal antibody wasused to determine the level of expression of the hybrid core pre-S genein the transformed attenuated S. typhi carrier strains derived fromMGN-1191 and MGN-1256. The expression of the hybrid HBcAg-pre-S antigenin ΔphoPQ Δasd mutant S. typhi strains was determined as follows.Proteins from whole bacterial cell lysates after overnight culture wereseparated using 12% sodium dodecyl sulfate (SDS-12%), polyacrylamide gelelectrophoresis (PAGE) and stained with Coomassie brilliant blue.Results are shown in FIG. 14. Three transformants of MGN-1191 and threetransformants of MGN-1256 were studied all of which showed a band at theposition of the recombinant antigen (see arrow in FIG. 14). The MGN-1191transformant #1 (lane 3) was designated χ8281 and the MGN-1256transformant #1 (lane 7) was designated χ8280. Both strains express theVi capsular antigen as determined by positive agglutination with Viantiserium (Difco).

In addition, the expression of the recombinant antigen was assessed byimmunoblotting. For immunoblotting, cells from overnight cultures weretaken up in 2× sample buffer and boiled for 10 minutes to lyse thecells. Proteins were separated by SDS-12% PAGE. The proteins weresubsequently transferred to nitrocellulose; incubated with monoclonalantibodies specific for HBV pre-S2; developed with peroxidase-coupledgoat anti-mouse immunoglobin G (IgG) (heavy and light chains) andvisualized on X-ray film (Kodak) after incubation with achemiluminescent substrate (ECL; Amersham). Results are shown in FIG.15. As was seen with Coomassie staining, the three transformants ofMGN-1191 including χ8281 (lane 3) and the three transformants ofMGN-1256 including χ8280 (lane 7) all showed a band at the position ofthe recombinant antigen (see arrow in FIG. 15).

For immunoscreening, the following procedure can be used. Bacterialcolonies are lifted onto nitrocellulose filters and lysed in 1% SDS for30 minutes at 70° C. Free binding sites on nitrocellulose are blocked by10% horse serum in Tris-HCl-buffered saline. Subsequently, immunoscreensare treated like immunoblots and secondary goat anti-mouse IgG (heavyand light chains) is visualized with nitrobluetetrazolium-5-bromo-4-chloro-3-indolylphosphate toluidinium (Promega).

S. typhi rpoS⁺ strains expressing foreign antigens can also beconstructed using plasmid vectors with selectable markers other thanAsd⁺, including genes that confer resistance to drugs such as ampicillinand tetracycline. In addition, the recombinant vector encoding thedesired foreign antigen may be constructed using well known techniquessuch that the vector will insert into the bacterial chromosome byhomologous recombination or by transposition.

EXAMPLE 12

This example illustrates methods which can be used in constructingrecombinant attenuated vaccine strains that express foreign proteins soas to suppress, modulate, or augment immune responses in a beneficialway.

It is well known that live attenuated bacterial vaccines inducelong-lasting immunity by inducing T helper lymphocyte memory functions.S. typhimurium infection of mice leads predominantly to a Th-1 type ofresponse although a Th-2 response with production of SIgA in mucosalsecretions and serum antibodies against Salmonella and against foreignexpressed antigens is also induced. IL-10 can be detected at levelsindicating the occurrence of the Th-2 response (Van Cott et al., J.Immunol. 156:1504-1514, 1996). It also known that the recombinantattenuated S. typhimurium vaccine can also induce a CTL responseinvolving CD-8⁺ cells against a foreign antigen (Sadoff et al., Science240:336, 1988). In many cases, however, it would be desirable if arecombinant attenuated Salmonella vaccine elicits predominantly a Th-2type of response to enhance mucosal immunity by the production of SIgAand a cellular memory response for that SIgA production. The lymphokinesIL-4 and IL-5 when produced, potentiate such a Th-2 response. On theother hand, it is desirable in other instances to maximize the abilityof the recombinant attenuated Salmonella to induce a Th-1 type ofresponse which might be particularly important in providing protectiveimmunity against a facultative or obligate intracellular parasite whoseantigens are expressed by the recombinant attenuated Salmonella vaccine.Shifting the immune response to a predominantly Th-1 or to a Th-2 typeof response can be achieved in part by expressing lymphokines viarecombinant attenuated Salmonella strains. Thus, we have constructedSalmonella strains expressing IL-2 which enhances the Th-1 type ofresponse and also potentiates a CTL response which is important indesigning attenuated Salmonella vaccines to be protective in combatingcertain types of cancer (Saltzman et al., Cancer BioTher. Radiol. Pharm.11:145-153, 1996; Saltzman et al., J. Pediatric Surg. 32:301-306, 1997).Generating the Salmonella to induce a predominant Th-2 response can beachieved by causing the strains to express IL-4 and IL-5 as has beendone for the latter lymphokine by Whittle et al. (1997, J. Med.Microbiol. 46:1029-1038). IL-4 has been expressed by a recombinant aroAattenuated Salmonella vaccine strain but was not effective since it wasnot secreted (Denich et al., Infect. Immun. 61:4818-4827, 1993). Methodssuch as described by Hahn et al. (FEMS Immunol. Med. Microbiol.20:111-119, 1998) are now available to succeed in such secretedexpression of lymphokines by attenuated Salmonella. It is also possibleto coexpress peptides such as factor P which is reported to stimulatethe secretion of SIgA. Genes for cDNAs have been obtained which specifymany different lymphokines, cytokines and other peptide or proteinmolecules which act to modulate the immune response. It is anticipatedthat these peptides or proteins could be coexpressed by recombinantattenuated Salmonella vaccine strains expressing some antigen from aparticular pathogen or from a tumor cell line or some other moleculethat was targeted for an immune response that would induce an immuneresponse to protect against an infectious disease or to therapeuticallycorrect against a systemic disease of the immunized human. Thus IL-6 hasbeen expressed and in some cases secreted by recombinant attenuatedSalmonella (Dunstan et al., Infect Immun 64:2730-2736, 1996; Hahn etal., FEMS Immunol Med Microbiol 20:111-119, 1998). The genes for murinemacrophage inhibitory factor (MIF), IL-2, IFN-γ or TNF-α wereindividually cloned and expressed by recombinant attenuated Salmonellato alter immune responses against Leishmania major infection (Xu et al.,J. Immunol. 160:1285-1289, 1998). TGF-β has also been expressed inrecombinant attenuated Salmonella vaccine strains to decrease theinflammatory response by inhibiting endogenous synthesis of IL-2 andINF-γ but enhancing synthesis of IL-10 (Ianaro et al., Immunology84:8-15, 1995). Based on data presented in preceding examples, it isevident that recombinant attenuated Salmonella vaccines of the RpoS⁺phenotype will be superior to vaccine strains of an RpoS⁻ phenotype inexpressing cytokines and other immunoactive molecules to suppress,enhance and/or modulate the immune response in a desired way.

EXAMPLE 13

This example illustrates methods which can be used in constructingrecombinant attenuated vaccine strains to combat cancer by supressingtumor growth, by enhancing the immunized individuals immune system toeliminate tumor cells and/or by inducing an immune response against atumor-specific antigen.

As stated in Example 12 above, we have constructed Salmonella strainsexpressing IL-2 which enhances the Th-1 type of response and alsopotentiates a CTL response which has been effective in decreasingmetastases by murine adenocarcinoma MC-38 (Saltzman et al., 1996:Saltzman et al, 1997). Based on results presented, it is evident that arecombinant attenuated Salmonella vaccine designed to supress tumorgrowth and spread would be more efficacious if displaying an RpoS⁺phenotype rather than a RpoS⁻ phenotype.

It is known that Salmonella will seek out and partially destroy solidtumors following infection of a tumor-bearing individual. (Pawelek etal., Cancer Res 57:4537-4544, 1997). Such wild-type Salmonellaultimately kill the host as well as destroy the tumor. It is, therefore,necessary to attenuate the Salmonella and also to modify it to eliminatethe inflamatory response resulting from the induction of TNFα by thelipid A moiety of LPS. This has been accomplished by using apurine-requirement mutant that is attenuated with an inactivated msbBgene which renders the lipid A non-inflamatory (Low et al, NatureBiotechnol. 17:37-41, 1999). Such strains can be further modified byintroduction of a Δasd mutation and endowed with an Asd⁺ plasmid vectorspecifying an enzyme that converts a non-toxic prodrug into ananti-tumor drug within the tumor to further enhance the rate of tumordestruction (see, for example, WO9913053). Based on results presented,it is evident that a recombinant attenuated Salmonella vaccine designedto supress tumor growth and spread would be more efficacious ifdisplaying an RpoS⁺ phenotype rather than a RpoS⁻ phenotype.

The attenuated S. typhi strains with the balanced-lethal host-vectorsystem as described in Example 11 can also be used to expresstumor-specific antigens to create a therapeutic anti-cancer vaccine.Such vaccines would thus be endowed with the ability to express aspecific tumor-specific antigen often fused to a T-cell epitope toenhance induction of a CTL response. Such a vaccine could also beenhanced by secreting IL-2 and rendered less inflamatory by introducinga msbB mutation or one of similar effect in depressing the inflamatoryeffect of lipid A. Based on results presented, it is evident that arecombinant attenuated Salmonella vaccine designed to supress tumorgrowth and spread would be more efficacious if displaying an RpoS⁺phenotype rather than a RpoS⁻ phenotype.

EXAMPLE 14

This Example illustrates the use of attenuated RpoS⁺ Salmonella strainshaving high immunogenicity and low virulence, to express an autoantigenand to exert an antifertility benefit.

We have previously described how to use recombinant attenuatedSalmonella strains to express autoantigens so as to induce a state ofinfertility. This technology is disclosed in U.S. Pat. No. 5,656,488. Inaddition, Srinivasan et al. (Biol. Reproduct. 53:462-471, 1995)describes how to express a sperm-specific antigen from a recombinantattenuated Salmonella so as to induce antibodies against that spermantigen to effectively block the sperm-egg interaction in the mouse toinduce a state of infertility. The specific antigen was specified by amurine cDNA sequence and the recombinant Salmonella was able to inducein mice an immune response against that autoantigen. Similarly, Zhang etal. (1997, Biol. Reproduct 56:33-41) expressed in an attenuated S.typhimurium strain, the murine cDNA sequence encoding the zona pellucidaantigen, ZP-3. Mice immunized with Salmonella expressing thisautoantigen mounted an immune response to ZP-3. Antibodies to ZP-3coated the surface of ova in the ovary and effectively reduced theability of sperm to fertilize such eggs. It is also well known thatfusion of an autoantigen to a carrier antigen which is heterologous tothe host can lead to the induction of an immune response whichrecognizes the autoantigen as well as the heterologous carrier. In thecase of fertility, such immunization strategies could lead to thedevelopment of contraceptive vaccines.

EXAMPLE 15

This Example illustrates the use of an attenuated RpoS⁺ Salmonellavaccine engineered to express an allergen and to induce an immuneresponse to ameliorate the effect of that allergen.

Allergies to pollens, mold spores, insect parts, animal dander and thelike are due to the inhalation of air and/or ingestion of foodcontaining such allergens. The allergies that result are associated witha presence of IgE antibodies that bind to allergens which activate mastcells for release of histamines. As is well known, desensitizationagainst allergens can be achieved by repetitive parenteral immunizationof extracts containing the allergen. Likewise, it is known that oralingestion of raw honey containing pollens can be used to effectivelyinduce a state of tolerance against those allergens. Oral ingestion withsuch allergens can on the one hand induce an SIgA response that couldblock the ability of allergens to react with IgE and mast cells or ifadministered in sufficient quantity could serve to suppress thesynthesis of IgE antibodies, that is to induce tolerance. Since thespecific allergenic molecule in many allergens has been identified andthe cDNA cloned to obtain the nucleotide sequence specifying theallergen, it is now possible to genetically engineer heterologous hostcells to express the allergen (see for example, Valenta et al, Allergy53:552-561. 1998; Olsson et al., Clin. Exp. Allergy 28:984-991. 1998;Soldatova et al., J. Allergy Clin. Immunol. 101:691-698, 1998; Asturiaset al, Clin. Exp. Allergy 27:1307-1313; Twardosz et al, Biochem.Biophys. Res. Comm. 239:197-204, 1997). Accordingly, the attenuatedRpoS⁺ Salmonella of the present invention can be engineered to expressan allergen, possibly in a modified immunogenic but nonallergenic formto induce a state of tolerance or to actively promote the production ofSIgA against the allergen. The RpoS⁺ attenuated Salmonella describedherein have been shown to be effective in eliciting immune responsesand, hence, it follows that use of such RpoS⁺ Salmonella to expressmodified allergens would be likely to be effective in ameliorating theconsequences of exposure of humans to allergens by inhalation oringestion.

EXAMPLE 16

This example illustrates a procedure that can be used for testing thesafety, immunogenicity and efficacy of live oral vaccines comprisingrecombinant attenuated rpoS⁺ S. typhi carrier strains which express adesired foreign antigen.

Strains tested are attenuated derivatives of ISP1820 and ISPI822 orattenuated derivatives of Ty2 strain χ8438 (Table 1) containing arecombinant rpoS gene.

The Individuals Studied: The individuals studied are volunteers who arehealthy adult humans age 18-40 years of either sex. The prospectivevolunteers are screened before the study. The inclusion criteriaincludes:

1. general good health;

2. evaluation of medical history;

3. normal and regular bowel habits;

4. normal physical examination;

5. normal laboratory findings including:

normal urinalysis,

normal complete blood count and differential,

normal blood chemistries (SGPT, alkaline phosphatase, BUN, creatinine,fasting blood glucose),

negative ELISA for HIV-1

negative pregnancy test (females);

6. able to understand and comply with required procedures including thepractice of good hygiene, maintenance of daily logs and willingness toundergo stool collection.

The exclusion criteria includes:

1. history of gall bladder disease;

2. gastric achlorhydria (frequent antacid, H² blocker or B₁₂ usage);

3. history of immunodeficiency;

4. positive pregnancy test (females)

5. medical, psychiatric or occupational condition which would precludecompliance with protocol;

6. diarrheal illness;

7. history of antibiotic therapy within 7 days prior to immunization;

8. history of drug allergy or serious adverse reaction to vaccines.

Volunteers are screened and informed written consent is obtained.

Study Design: Groups of 5 or 6 volunteers are studied for each strainand dose. In the first group of volunteers, the subjects will receive asingle dose of 10⁵ CFU of the attenuated vaccine. If this group developsno clinical symptoms of disease, an escalation in dose will proceed insubsequent groups to establish the maximal safe and minimal immunogenicdose. Subsequent groups will receive 10⁶ CFU or greater doses up to amaximal dose of 10⁹ CFU.

Preparation of the vaccine inocula: Stock cultures of the S. typhicandidate vaccine strains are stored as a cell suspension in 1%bactopeptone (Difco) containing 5% glycerol at −70° C. To make aninoculum of the strain, the suspension is thawed and then diluted to theappropriate CFU/ml for the particular dose.

Inoculation of Volunteers: On the day of inoculation of volunteers,blood, urine and stool samples are obtained and baseline values forclinical laboratory parameters are determined. In addition,immunoglobins are measured in serum and stool samples. The subjectsreceive nothing by mouth for 90 minutes before inoculation. Two grams ofNaHCO₃ are dissolved in 5 ounces of distilled water. The subjects willdrink 4 ounces of the bicarbonate water and one minute later thesubjects will ingest the vaccine suspended in the remaining one ounce ofbicarbonate water. Subjects will take no food or water for 90 minutesafter inoculation.

Clinical monitoring of volunteers: The volunteers are followed asinpatients for a minimum of two weeks and thereafter as outpatients upto a total of four weeks. During this period observations are made forany adverse effects including but not limited to fever, headache,chills, vomiting, diarrhea and abdominal pain. Blood and stool samplesare obtained during the testing period and cultures and antibodydeterminations are done. In addition, PCR for vaccine strain is done onserum. Any volunteer who develops a temperature of 100.8° F. at any timeduring the study will have stool samples and blood drawn for culture; ifthe temperature remains elevated for 12 hours and/or blood culture ispositive, a 10 day course of oral antibiotics will be given.

Procedures for Specimen Collection.

Stool Specimens: A record will be kept of the number, consistency anddescription of all stools passed by volunteers for 14 days postvaccination. Stool volume will be measured and the stool will be gradedon a 5 point system:

Grade 1—firm stool (normal)

Grade 2—soft stool (normal)

Grade 3—thick liquid (abnormal)

Grade 4—opaque watery (abnormal)

Grade 5—rice water (abnormal)

Stool cultures will be performed on a sample of stool (or rectal swab ifstool was not passed) each day on consecutive days for Salmonella untilnegative times one.

Phlebotomy: Serum (20 ml blood) will be collected for prescreeningevaluation. Serum for antibody (10 ml blood) determinations will beobtained on days 0, 7, 14 and 28. Heparinized blood for lymphocyteseparation (30 ml) for antibody-secreting cell assays by ELISPOT will becollected on days 0, 7, 14 and 28 on a subset of volunteers. The subsetwill consist of 2 volunteers in groups 3, 4 and 5. Volunteers will beselected randomly by the computer. Blood (10 ml) will be obtained forculture on each day until negative during the post immunizationobservation period to detect viable vaccine organisms by bothconventional culture and PCR. In total, no more than 450 ml of bloodwill be collected from any volunteer during any 2 month period. Bacteriain positive blood cultures will be evaluated for conformity to thegenotype/phenotype of the vaccine strain.

Bacteriology: Stools and rectal swabs will be inoculated intoselenite-cystine broth. Stools must be processed within 48 hours. Afterovernight incubation at 37° C., subcultures will be made onto XLT-4agar. Colonies which appear consistent with Salmonella will be processedthrough API-20 system of identification and confirmation made by aagglutenation with S. typhi O, H, and Vi antisera. These isolates willbe saved at −70° C. in 5% glycerol-1% peptone for further analysis(e.g., for the presence of plasmids, for absence or presence of specificDNA sequences using PCR, or for Southern blotting with gene probes forcloned genes).

Blood cultures (10 ml) will be inoculated in 50 ml Septacheck bottles.Positive cultures are analyzed and saved as described above.

Immunology: Sera specimens will be tested for IgA, IgM and IgG to S.typhi O, H and Vi antigens measured by ELISA. H antibody will also bemeasured by Widal tube agglutination using S. virginia as antigen (S.manhatten also shares the identical flagellar antigen as S. typhi butnot somatic antigen). Peripheral blood mononuclear cells will becollected and separated for antibody secreting cell (ASC) assaysemploying ELISPOT for cells producing antibody to Salmonella antigens.Lymphocytes that secrete IgG, IgA, or IgM against S. typhi O, H, or Viantigens will be measured.

PCR: The Salmonella invA gene segment will be amplified by polymerasechain reaction to confirm the presence or absence of Salmonella typhi inblood specimens. The invA sequence is unique to Salmonella (Galan andCurtiss, 1991) and is diagnostic for the presence of invasive Salmonellaby PCR methods (Rahn et al., 1992).

Excretion of the Vaccine Strain: It is expected that excretion of thevaccine strain would cease within 1 week after a dose of vaccine. Ifexcretion continues for 7 or more days, the volunteer who continues toexcrete is given a dose of ciprofloxacin (700 mg every 12 hours).Negative cultures for a ≧2 consecutive days are required for discharge.

EXAMPLE 17

This Example illustrates the potential use of attenuated RpoS⁺Salmonella enterica of various serotypes for intranasal administrationto elicit superior mucosal and systemic immune responses. Such candidatevaccines can also be administered orally, conjuntivally, or rectally.

The Salmonella enterica serotypes are, preferably, attenuated with knownattenuation approaches such as by generating deletion mutations in a pabgene, a pur gene, an aro gene, asd, a dap gene, nadA, pncB, galE, pmi,fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc, poxA,galU, metL, metH, mviA, sodC, recA, ssrA, ssrB, sirA, sirB, sirC, inv,hilA, hilC, hilD, rpoE, flgm, tonB, slyA, or in a combination of thesegenes. Furthermore, the microbes would have an RpoS⁺ phenotype asdetermined by the catalase test or the glycogen synthesis test asdescribed in Example 4. The RpoS⁺ attenuated Salmonella enterica strainscould be of the serotypes Typhi, Paratyphi A, Paratyphi B, Paratyphi C,Typhimurium, Enteritidis, Dublin, or Choleraesuis. In the wide hostrange S. enterica serotypes, Typhimurium and Enteritidis, and in themore host-adapted serotypes, Dublin and Choleraesuis, it is desirablethat they possess the Salmonella virulence plasmid which enhances theirimmunogenicity due to more rapid growth in intracellular in vivoenvironments (Gulig in Escherichia coli and Salmonella, Vol 2, F.Neidhardt et al., Editor, American Society for Microbiology, WashingtonD.C., pp. 2774-2787, 1996).

Study design for testing safety and efficacy in humans are as describedin Example 16 above except for the serotype of Salmonella to beadministered and the nasal route of immunization. Table 16 listsparental vaccine vector strains with differing serotypes and their testresults for catalase and glycogen synthesis to indicate the RpoS⁺phenotype.

TABLE 16 Determination of RpoS Phenotype of Bacterial Strains. GlycogenCatalase Bacterial species Accumulation/Boisynthesis^(a) Activity χ3246Salmonella − + choleraesuis χ3759 Salmonella + + enteritidis χ3841Salmonella + + infantis χ4952 Salmonella + + pullorum χ4821 Salmonellan.g. + dublin χ8274 Salmonella + + typhimurium 14028s χ8219 Salmonella +− paratyphi A χ8436 Salmonella + + paratyphi C χ8437 Salmonella + +sendai Shigella flexneri n.g. + 2a 2457T Shigella flexneri not tested +2a 15D ^(a)n.g. - no growth on Q-3 medium.

Note that S choleraesuis χ3246 is unable to synthesize glycogen,however, it tests as RpoS⁺ by the catalase test. S. paratyphi A, χ8219synthesizes glycogen indicating an RpoS⁺ phenotype, but lacks thecatalase regulated by the rpoS gene. Any of these strains can beattenuated by the methods described in Examples 2 and 3 and furthermodified with the asd mutation for use of an Asd⁺ vector encoding for aforeign antigen such as the pYA3167 specifying the hepatitis B viruscore pre-S1/preS-2 fusion as described in Example 11 and used in Example8. All of these strains display the RpoS⁺ phenotype.

The other difference between the procedures described in Example 16 isthe route of immunization. Intranasal immunization can be achieved byadministration of nose drops containing the vaccine strain at a suitabledose while the individual is lying prone with head turned back.Alternatively, intranasal immunization can be achieved by aerosolizationinto the nostrils with a nebulizer. The dose administered is determinedby the number of squirts.

Other routes of administration can also be tested. For example, rectalimmunization can be achieved by the procedures described byNardelli-Haefliger et al. (Infect Immun, 1996). Intraconjunctivalimmunization can be achieved by administration of eye drops. All of themonitoring and well being of immunized subjects and for the elicitationof appropriate immune responses are as described in Example 16.

EXAMPLE 18

This example illustrates methods for preparation of RpoS⁺ Salmonella,Shigella/Escherichia and Salmonella/Escherichia hybrids for use indelivering DNA vaccine vectors to a human.

Circular plasmid DNA encoding antigens of various pathogens can beintroduced into animal hosts to stimulate the induction of immunity tothe pathogen from which the antigen gene was derived (Ullmer et al., ASMNews 62:476-479, 1996; Ullmer et al., Curr. Opin. Immunol 8:531-536,1996; Whalen, Emerg. Infect. Dis. 2:168-175, 1996; Robinson, Vaccine15:785-787, 1997). DNA vaccines make use of expression systems such thatthe genetic information specifying the antigen of some pathogen isexpressed by the immunized hosts using host machinery for transcriptionand translation. Initially, DNA vaccines were administered by injectioninto muscle tissue, but other injection sites have also been used. Mostrecently, DNA vaccines have been administered using particle guns toaccelerate entry of DNA-coated gold beads into skin or mucosal tissues.The DNA vaccine vbectors are propagated in and isolated from recombinantE. coli strains grown in fermentors.

Sizemore et al. (Science, 270:299-302, 1995; Vaccine 15:804-807, 1997)described the use of Shigella flexneri 2a strain 15D with a Δasdmutation that harbored a DNA vaccine vector engineered to express E.coli β-galactosidase. The Shigella strain was attenuated due to the Δasdmutation which causes death due to absence of diaminopimelic acid uponinvasion into eukaryotic cells. The strain was able to deliver the DNAvaccine vector intracellularly after attachment to, invasion into andlysis within the cytoplasm of eukaryotic cells in culture or withinimmunized mice. More recently, others have used S. typhimurium strainspossessing a DNA vaccine vector and caused to lyse by spontaneous means(Powell et al., WO96/34631, 1996; Pasenal et al., Behring. Inst. Mitt.98:143-152, 1997; Darji et al., Cell 91:765-775, 1997). In cases inwhich lysis was spontaneous, it was necessary that the bacterial strainpossess one or more deletion mutations rendering the strain attenuated.Shigella, Salmonella and invasive E. coli are known to have a muchenhanced ability to attach to and invade M cells overlying the GALTrather than to attach to and invade intestinal epithelial cells(enterocytes). Delivery of foreign antigens or the production of foreignantigens within the NALT, BALT, CALT and GALT which all have an M celllayer leads to induction of mucosal immune responses as well as systemicimmunity. Because mucosal immune responses are protective against thevast majority of infectious disease agents that colonize on or invadethrough a mucosal surface, it would be expected that DNA vaccine vectorscould thus be delivered by RpoS⁺ Salmonella, Shigella, Escherichia orhybrids between any two of these genera. These microbes would have asuperior ability to attach to and invade the M cells overlying thelymphoid tissues of the NALT, CALT, BALT and GALT. Because both oral andintranasal immunization with RpoS⁺ microbes increase the immuneresponse, it would be expected that attenuated bacterial DNA vaccinevector strains displaying an RpoS⁺ phenotype will give an increasedimmune response when administered intranasally or perorally andpresumably by other routes that stimulate mucosal immune responses.

We have used derivatives of the DNA vaccine vector pCMVβ to expressforeign antigens. pCMVβ possesses the pUC origin of replication forpropagation in E. coli, a β-lactamase gene to confer resistance toampicillin, promoters and enhancers from CMV and SV40 viruses and anSV40 sequence to achieve polyadenylation of the transcribed mRNA(MacGregor et al., Nucleic Acid Res. 17:1265, 1989). pCMVβ contains thecoding sequence for E. coli β-galactosidase which has been used as atest antigen in several studies. The lacZ gene encoding β-galactosidasecan be easily removed with substitution of DNA encoding a diversity ofantigens, especially from viral, fungal and parasitic pathogens. Sinceintroducing antibiotic resistance genes as parts of vaccines intoimmunized animal and human hosts continues to be a concern, we havesubstituted the S. typhimurium asd gene for the ampicillin-resistancegene in pCMVβ to yield pCMVβ-asd (FIG. 16). This enables the use of anE. coli host that has a Aasd mutation to yield a balanced-lethalhost-vector system that can be propagated in the fermentor in theabsence of added costly antibiotics that could also potentiallycontaminate the purified DNA vaccine vector. Furthermore, the S.typhimurium asd gene possesses two natural CpG sequences (Kreig, J. Lab.Clin. Med 128:128-133, 1996) that strongly enhance the immunogenicity ofthe DNA vaccine vector. Such sequences are absent in thekanamycin-resistance gene that is now often used in lieu of theampicillin-resistance gene in DNA vaccines. The use of the S.typhimurium asd gene in such DNA vaccine vectors is described in U.S.Pat. No. 5,840,483.

Further refinement of this technology to improve efficacy would be tohave the attenuated bacteria release into the cytoplasm of antigenpresenting cells within the immunized individual, mRNA copies of genespresent in the DNA vaccine vector so that the mRNA would be directlytranslated into a protein product within the cytoplasm of the immunizedhost's cells. This could greatly enhance the efficiency of vaccinedelivery since with traditional delivery of DNA vaccines, the DNAvaccine vector must migrate to the nucleus to permit transcription whicheven then might not occur in all cells, and then have the mRNA transitto the cytoplasm for translation into a protein product to stimulate animmune response. Since RpoS⁺ strains are more efficient than RpoS⁻strain in antigen delivery, it is expected that they would also be moreefficient in delivery of nucleic acid, either DNA or RNA.

Commercial Utility

The bacterial strain,s provided herein are directly and indirectlysuitable for production of immunogenic compositions, including vaccines,to prevent diseases caused by various bacterial, viral, fungal protazoalpathogenes. These carrier bacterial strains which can be, for example,S. typhi strains or other Enterobacteriaceae, all have an RpoS⁺phenotype, and can serve as carriers for delivering to target tissues,heterologous proteins or nucleic acid molecules for expression of geneproducts. Examples of gene products deliverable by the microbes of theinvention include but are not limited to: antigens, which can be from ahuman pathogen, or, for use in autoimmune applications, from the humanitself, such as, for example, a gamete-specific antigen; enzymes thatcan synthesize antigens such as polysaccharides, lipoproteins,glycoproteins, and glycolipids; allergens of the human; immunoregulatorymolecules; hormones; and pharmacologically active polypeptides. Themicrobes are not only attenuated, but also show high immunogenicitybecause of an improved ability to colonize lymphoid tissue compared topreviously used recombinant attenuated bacteria. The present strains areuseful as carrier microorganisms for the production of expressionproducts encoded on recombinant genes in the bacterial cells. Inaddition, the strains which can be. used with enhanced safety andimproved immunogenicity are highly effective in the production ofantibodies against recombinant antigens which can be expressed in theattenuated, immunogenic bacteria.

Deposit

The following strains and plasmid are on deposit under the terms of theBudapest Treaty, with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. The accession number indicated wasassigned after successful viability testing, and the requisite fees werepaid. Access to the cultures and plasmid will be available duringpendency of the patent application to one determined by the Commissionerto be entitled thereto under 37 CFR 1.14 and 35 USC 122. All restrictionon availability of the cultures and plasmid to the public will beirrevocably removed upon the granting of a patent based upon theapplication. Moreover, the designated deposits will be maintained for aperiod of thirty (30) years from the date of deposit, or for five (5)years after the last request for the deposit, or for the enforceablelife of the U.S. patent, whichever is longer. Should a culture orplasmid become nonviable or be inadvertently destroyed, or, in the caseof plasmid-containing strains, lose its plasmid, it will be replacedwith a viable culture. The deposited materials mentioned herein areintended for convenience only, and are not required to practice thepresent invention in view of the description herein, and in addition,these materials are incorporated herein by reference.

Deposit Deposit Date ATCC No. Strains MGN-1191 November 14, 1997 202054MGN-1256 November 14, 1997 202053 χ8280 November 14, 1997 202055 χ8281November 14, 1997 202056 χ8438 November 18, 1998 202182 Plasmid pYA3433November 14, 1997 209462

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

What is claimed is:
 1. A method for producing, from a parent bacteriastrain, a carrier bacteria for the delivery of a desired gene product toa human comprising generating a strain of bacteria comprsising (a) arecombinant rpoS⁺ gene; (b) one or more inactivating mutations whichrender said bacteria attenuated; and (c) a second recombinant geneencoding the desired gene product, wherein said carrier bacteriaexpresses a higher level of RpoS gene product than said parent bacteriastrain and wherein said higher level of RpoS gene product confers uponthe carrier bacteria high immunogenicity relative to said parentbacteria strain.
 2. The method of claim 1, said bacteria lacks afunctional chromosomal rpoS⁺ gene.
 3. The method according to claim 1wherein the bacteria is a strain of Salmonella.
 4. The method accordingto claim 3 wherein the Salmonella is a strain of S. typhi.
 5. The methodaccording to claim 4 wherein the one or more inactivating mutations arein a gene selected from the group consisting of a pab gene, a pur gene,an aro gene, asd, a dap gene, nadA, pncB, galE, pmi, fur, rpsL, ompR,htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc, poxA, galU, metL, metH,mviA, sodC, recA, ssrA, ssrB, sirA, sirB, sirC, inv, hilA, hilC, hilD,rpoE, flgM, tonB, and slyA.
 6. The method according to claim 5 whereinthe second recombinant gene encodes a gene product from a pathogen tosaid human.
 7. The method according to claim 6 wherein the pathogen is avirus, bacterium, protozoan, parasite or fungus.
 8. A carrier bacteriafor the delivery of a desired gene product to a human produced accordingto the method of claim
 1. 9. The carrier bacteria of claim 8, whereinsaid bacteria lacks a functional chromosomal rpoS+ gene.
 10. A carrierbacteria according to claim 8 wherein the bacteria is a Salmonella. 11.A carrier bacteria according to claim 10 wherein the Salmonella is an S.typhi.
 12. The carrier bacteria according to claim 11 wherein the one ormore inactivating mutations are in a gene selected from the groupconsisting of a pab gene, a pur gene, an aro gene, asd, a dap gene,nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, dam,phoP, phoQ, rfc, poxA, galU, metL, metH, mviA, sodC, recA, ssrA, ssrB,sirA, sirB, sirC, inv, hilA, hilC, hilD, rpoE, flgM, tonB, and slyA. 13.The carrier bacteria according to claim 12 wherein the secondrecombinant gene encodes a gene product from a pathogen to said human.14. The carrier microbe according to claim 13 wherein the pathogen is avirus, bacterium, protozoan, parasite or fungus.
 15. A composition forimmunization of a human comprising a carrier bacteria according to claim3.
 16. The composition of claim 15, wherein said bacteria lacks afunctional chromosomal rpoS+ gene.
 17. The composition according toclaim 15 wherein the bacteria is a Salmonella.
 18. The compositionaccording to claim 17 whreein the Salmonella is an S. typhi.
 19. Thecomposition according to claim 18 wherein the one or more inactivatingmutations are in a gene selected from the group consisting of a pabgene, a pur gene, an aro gene, asd, a dap gene, nadA, pncB, galE, pmi,fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc, poxA,galU, metL, metH, mviA, sodC, recA, ssrA, ssrB, sirA, sirB, sirC, inv,hilA, hilC, hilD, rpoE, flgM, tonB,and slyA.
 20. The compositionaccording to claim 19 wherein the second recombinant gene encodes a geneproduct from a pathogen to said human.
 21. The composition according toclaim 20 wherein the pathogen is a virus, bacterium, protozoan, parasiteor fungus.
 22. The composition according to claim 18 wherein saidattenuated strain of S. typhi is in a pharmaceutically acceptablecarrier.
 23. A genetically engineered bacterial cell, wherein saidgenetically engineered bacterial cell (a) is produced from a parentbacterial cell, (b) is a live attenuated strain of bacteria, (c) has arecombinant rpoS⁺ gene, (d) has one or more inactivating mutations whichrender said bacteria attenuated and (e) has a second recombinant geneencoding a desired gene product, and wherein the genetically engineeredbacterial cell expresses a higher level of RpoS gene product than saidparent bacteria cell and wherein said higher level of RpoS gene productconfers upon the genetically engineered bacterial cell highimmunogenicity relative to said parent bacteria strain.
 24. Thegenetically engineered bacterial cell of claim 23, wherein saidbacterial cell lacks a functional chromosomal rpoS+ gene.
 25. Thegenetically engineered bacterial cell according to claim 23 wherein thestrain of bacteria is a strain of Salmonella.
 26. The geneticallyengineered bacterial cell according to claim 25 wherein the strain ofSalmonella is a strain of S. typhi.
 27. The genetically engineeredbacterial cell according to claim 26 wherein the one or moreinactivating mutations are in a gene selected from the group consistingof a pab gene, a pur gene, an aro gene, asd, a dap gene, nadA, pncB,galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, dam, phoP, phoQ,rfc, poxA, galU, metL, metH, mviA, sodC, recA, ssrA, ssrB, sirA, sirB,sirC, inv, hilA, hilC, hilD, rpoE, flgM, tonB, and slyA.
 28. Thegenetically engineered bacterial cell according to claim 27 wherein thesecond recombinant gene encodes a gene product from a pathogen to saidhuman.
 29. The genetically engineered bacterial cell according to claim28 wherein the pathogen is a virus, bacterium, protozoan, parasite orfungus.
 30. A method for preparing an immunogenic composition, themethod comprising mixing the genetically engineered bacterial cellaccording to claim 23 with a pharmaceutically acceptable carrier. 31.The method of claim 30, wherein said bacterial cell lacks a functionalchromosomal rpoS+ gene.